Methods of therapeutic treatment of eyes

ABSTRACT

Provided are electrokinetically-altered aqueous fluids (e.g., gas-enriched electrokinetic fluids) comprising an ionic aqueous solution of charge-stabilized oxygen-containing nanostructures in an amount sufficient to provide modulation of at least one of cellular membrane potential and cellular membrane conductivity, and therapeutic compositions and methods for use in treating an irritation, infection or inflammatory eye condition, comprising administering to, by contacting the eye of a subject in need thereof a therapeutically effective amount of an electrokinetically-altered aqueous fluid. The electrokinetically-altered fluids or therapeutic compositions and methods include electrokinetically-altered ioinic aqueous fluids optionally in combination with other therapeutic agents. Other embodiments include particular routes of administration or formulations for the electrokinetically-altered fluids (e.g., electrokinetically-altered gas-enriched fluids) and therapeutic compositions for use in treating eye conditions. Certain embodiments relate to cosmetic and/or therapeutic fluids and/or methods of treatment utilizing the fluids to treat a cosmetic and/or therapeutic symptom related to eye conditions and/or diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 11/924,601 filedOct. 25, 2007, which claims priority to U.S. Provisional ApplicationNos. 60/862,953 filed Oct. 25, 2006, 60/862,959 filed Oct. 25, 2006,60/862,955 filed Oct. 25, 2006, 60/862,904 filed Oct. 25, 2006,60/982,387 filed Oct. 24, 2007, and additionally claims priority to U.S.Provisional Application No. 61/048,357 filed Apr. 28, 2008, all of whichare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

Certain embodiments disclosed herein relate generally to the field ofeye care and treatment, and more particularly to compositions andmethods for treating irritation, infection or inflammatory eye conditionor disease of a subject comprising administering a therapeuticcomposition comprising at least one electrokinetically generated fluid(including gas-enriched (e.g., oxygen enriched) electrokineticallygenerated fluids) as disclosed herein for modulation of at least one ofcellular membrane potential and cellular membrane conductivity,modulating intracellular signal transduction by modulation of at leastone of cellular membranes, membrane potential, and membrane proteinssuch as membrane receptors, including but not limited to G proteincoupled receptors. Particular aspects relate to treating at least onedisease or condition or symptom thereof associated with eye care andtreatment.

BACKGROUND OF THE INVENTION

The eye of an animal, particularly a human, has an outer covering calleda cornea, which refracts light rays as a first step in the process ofthe animal visualizing an object. The surface of the animal eye,particularly the human eye, is normally bathed by a tear film that issecreted by tiny glands around the eye. The tear film is primarilycomposed of three layers: mucous, water, and oil. The mucous layer isclosest to the eye organ and serves as an anchor for the tear film toadhere to the eye. The middle layer is an aqueous layer, and the outeroil layer seals the tear film and prevents evaporation. The tear filmalso contains various nutritive and protective proteins and peptides.The tear film serves several purposes: it keeps the eye moist, creates asmooth surface for light to pass through the eye, nourishes the front ofthe eye, and provides protection from injury and infection.

The eye, particularly the cornea, can become dry, irritated, or damagedby trauma, disease, natural aging, environmental factors (such aspollutants and allergens), or by wearing contact lenses. In some cases,individuals may experience burning, a feeling of dryness, scratchiness,itchiness, or persistent irritation or inflammation. This irritation canlead to infection and/or visual impairment if not treated properly.

Red, irritated, and scratchy eyes are a common ophthalmologicaloccurrence affecting millions of Americans each year. In some cases,individuals may experience burning, a feeling of dryness, scratchiness,itchiness, or persistent irritation or inflammation such as is oftencaused by particles that are lodged between the eye lid and the eyesurface. This irritation can lead to infection and/or visual impairmentif not treated properly.

Dry Eye.

Keratoconjunctivitis sicca (KCS), more commonly known as dry eye,affects more than 40 million Americans and countless more worldwide. KCSis an under-diagnosed disease which can produce symptoms of oculardryness, discomfort, and in severe cases, visual impairment. KCS is theresult of changes in tear film dynamics leading to one of twoconditions: a decrease in aqueous tear production or an increase in tearevaporation. Both conditions create a state of hyperosmolar stress,which leads to an upregulation of several pro-inflammatory mediators andsubsequent ocular surface damage. The mainstay for treatment of dry eyehas been the use of over-the-counter artificial tear products. Morerecent advances include the additional use of topical anti-inflammatoryagents to reduce inflammation and increase basal tear production.

Allergy Eye.

Allergic conjunctivitis, or allergy eye, is another fairly common ocularcondition. Allergic conjunctivitis may be seasonal or perennial, and maybe triggered by any number of agents. Several common treatments forallergy eye presently include antihistamines, mast cell stabilizers,nonsteroidal anti-inflammatory drugs, or corticosteroids.

Allergic conjunctivitis has an immune-mediated inflammatory componsent.In susceptible individuals, initial exposure to the causative allergenstimulates production of antigen-specific antibodies (such as IgE),which bind to the surface of the mast cells and basophils in theconjunctiva. Typical clinical manifestations of allergic conjunctivitisinclude erythema, edema, and itching. Presently, eye drops approved forthe treatment of allergic conjunctivitis are aimed at preventing ortreating allergic signs and symptoms. Allergy eye occurs in phases orstages, such that several hours following the initial mast cell mediatedevent, the clinical symptoms may worsen during the so-called late phasereaction. The late phase reaction is characterized by an influx ofinflammatory cells, including neutrophils, lymphocytes, and eosinophils.This results in the release of a second wave of inflammatory mediatorssuch as leukotrienes, prostaglandins, and platelet activating factor(PAF).

Prior Approaches.

The most common approach to treatment of irritated, allergic, and/or dryeyes has been to supplement the natural ocular tear film using so-calledartificial tears instilled throughout the day. Examples of artificialtears include buffered, isotonic saline solutions, and/or aqueoussolutions containing water soluble polymers that render the solutionsmore viscous and thus less easily shed by the eye. Tear reconstitutionis also attempted by providing one or more components of the tear filmsuch as phospholipids and oils. Phospholipid compositions have beenshown to be useful in treating dry eye; see, e.g., McCulley and Shine,Tear film structure and dry eye, Contactologia, volume 20(4), pages145-49 (1998); and Shine and McCulley, Keratoconjunctivitis siccaassociated with meibomian secretion polar lipid abnormality, Archives ofOphth., volume 116(7), pages 849-52 (1998). Examples of phospholipidcompositions for the treatment of dry eye are disclosed in U.S. Pat. No.4,131,651 (Shah et al.), U.S. Pat. No. 4,370,325 (Packman), U.S. Pat.No. 4,409,205 (Shively), U.S. Pat. Nos. 4,744,980 and 4,883,658 (Holly),U.S. Pat. No. 4,914,088 (Glonek), U.S. Pat. No. 5,075,104 (Gressel etal.), U.S. Pat. No. 5,278,151 (Korb et al.), U.S. Pat. No. 5,294,607(Glonek et al.), U.S. Pat. No. 5,371,108 (Korb et al.) and U.S. Pat. No.5,578,586 (Glonek et al.).

Another approach involves the provision of lubricating substances inlieu of artificial tears. For example, U.S. Pat. No. 4,818,537 (Guo)discloses the use of a lubricating, liposome-based composition, and U.S.Pat. No. 5,800,807 (Hu et al.) discloses compositions containingglycerin and propylene glycol for treating eye irritation.

Other approaches include the use of ocular inserts that provide a tearsubstitute or stimulation of endogenous tear production. Such approacheshave been met with limited success, and provide merely temporary andminimal symptomatic relief.

There is, however, a profound need in the art for novel safe andeffective eye care treatments that allow for alleviation of eyeirritation and inflammation, and/or increases wetting of the eye.

SUMMARY OF THE INVENTION

Particular aspects provide methods for treating an irritation, infectionor inflammatory eye condition or disease of a subject, comprisingadministering to, by contacting the eye of a subject in need thereof atherapeutically effective amount of an electrokinetically-alteredaqueous fluid comprising an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide, upon contact ofa living cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity, wherein anirritation, infection or inflammatory eye condition or disease, or atleast one symptom thereof is treated. In further aspects, thecharge-stabilized oxygen-containing nanostructures are the majorcharge-stabilized gas-containing nanostructure species in the fluid.According to additional aspects, the percentage of dissolved oxygenmolecules present in the fluid as the charge-stabilizedoxygen-containing nanostructures is a percentage selected from the groupconsisting of greater than: 0.01%, 0.1%, 1%, 5%; 10%; 15%; 20%; 25%;30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; and95%.

In further aspects, the total dissolved oxygen is substantially presentin the charge-stabilized oxygen-containing nanostructures. In certainaspects, the charge-stabilized oxygen-containing nanostructuressubstantially have an average diameter of less than a size selected fromthe group consisting of: 90 nm; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30nm; 20 nm; 10 nm; and less than 5 nm. According to further aspects, theionic aqueous solution comprises a saline solution. In certain aspectsthe fluid is superoxygenated. In further aspects, the fluid comprises aform of solvated electrons. According to further aspects, theelectrokinetically altered aqueous fluid comprises exposure of the fluidto hydrodynamically-induced, localized electrokinetic effects. Incertain aspects, exposure to the localized electrokinetic effectscomprises exposure to at least one of voltage pulses and current pulses.In further aspects, the exposure of the fluid tohydrodynamically-induced, localized electrokinetic effects, comprisesexposure of the fluid to electrokinetic effect-inducing structuralfeatures of a device used to generate the fluid.

Particular aspects provide for the irritation, infection or inflammatoryeye condition or disease to comprise at least one selected from thegroup consisting of dry eye, allergy irritation, corneal irritation,bacterial infection, and cellular damage. In further aspects, theirritation, infection or inflammatory eye condition or disease eyecondition or disease comprises at least one of dry eye, allergyirritation, and corneal irritation. In certain aspects, the irritation,infection or inflammatory eye condition or disease eye condition ordisease comprises dry eye or allergy irritation.

According to certain aspects, at least one symptom of the irritation,infection or inflammatory eye condition or disease is related to atleast one condition selected from the group consisting of: dryeye-related irritation, dry eye-related cellular damage, and allergicinflammation. In further aspects, the electrokinetically altered aqueousfluid modulates localized or cellular levels of nitric oxide. Inadditional aspects, the electrokinetically altered aqueous fluidpromotes a localized decrease at the site of administration of at leastone cytokine selected from the group consisting of: IL-1-beta, IL-8,TNF-alpha, and TNF-beta.

According to certain aspects the invention further comprises asynergistic or non-synergistic inhibition or reduction in inflammationby simultaneously or adjunctively treating the subject with anotheranti-inflammatory or eye treatment agent. In further aspects, said otheranti-inflammatory agent comprises a steroid or glucocorticoid steroid.In additional aspects, the glucocorticoid steroid comprises Budesonideor an active derivative thereof.

According to certain aspects the invention further comprises combinationtherapy, wherein at least one additional therapeutic agent isadministered to the patient. In certain aspects, at least one additionaltherapeutic agent is selected from the group consisting of:anti-microbial agent, anti-inflammatory agent, pain reliever,anesthetic, vitamin, cytokine, adjuvant, preservative, salt, inhibitorsof MMPs including inhibitor of MMP-9 and MMP-2, short-acting β₂-agonistslong-acting β₂-agonists, anticholinergics, corticosteroids, systemiccorticosteroids, mast cell stabilizers, leukotriene modifiers,methylxanthines, β₂-agonists, albuterol, levalbuterol, pirbuterol,artformoterol, formoterol, salmeterol, anticholinergics includingipratropium and tritropium; corticosteroids including beclomethasone,budesonide, flunisolide, fluticasone, mometasone, triamcinolone,methyprednisolone, prednisolone, predinisone; leukotriene modifiersincluding montelukast, zafirlukast, and zileuton; mast cell stablizersincluding cromolyn and nedocromil; methylxanthines includingtheophylline; combination drugs including ipratropium and albuterol,fluticasone and salmeterol, budesonide and formoterol; antihistaminesincluding hydroxyzine, diphenhydramine, loratadine, cetirizine, andhydrocortisone; immune system modulating drugs including tacrolimus andpimecrolimus; cyclosporine; azathioprine; mycophenolatemofetil; andcombinations thereof. In additional aspects, at least one additionaltherapeutic agent is a TSLP and/or TSLPR antagonist. According tofurther aspects, the TSLP and/or TSLPR antagonist is selected from thegroup consisting of neutralizing antibodies specific for TSLP and theTSLP receptor, soluble TSLP receptor molecules, and TSLP receptor fusionproteins, including TSLPR-immunoglobulin Fc molecules or polypeptidesthat encode components of more than one receptor chain. In certainaspects, altering cellular membrane structure or function comprisesaltering of a conformation, ligand binding activity, or a catalyticactivity of a membrane associated protein. In additional aspects, themembrane associated protein comprises at least one selected from thegroup consisting of receptors, transmembrane receptors, ion channelproteins, intracellular attachment proteins, cellular adhesion proteins,integrins, etc. In further aspects, the transmembrane receptor comprisesa G-Protein Coupled Receptor (GPCR). In certain aspects, the G-ProteinCoupled Receptor (GPCR) interacts with a G protein α subunit. Accordingto further aspects, the G protein α subunit comprises at least oneselected from the group consisting of Gα_(s), Gα_(i), Gα_(q), and Gα₁₂.In additional aspects, at least one G protein α subunit is Gα_(q).

Particular aspects provide for modulating cellular membraneconductivity, which comprises modulating whole-cell conductance. Incertain aspects, modulating whole-cell conductance, comprises modulatingat least one linear or non-linear voltage-dependent contribution of thewhole-cell conductance. According to additional aspects, modulation ofat least one of cellular membrane potential and cellular membraneconductivity comprises modulation of a calcium-dependant cellularmessaging pathway or system. In further aspects, modulation of at leastone of cellular membrane potential and cellular membrane conductivitycomprises modulation of phospholipase C activity. In additional aspects,modulation of at least one of cellular membrane potential and cellularmembrane conductivity comprises modulation of adenylate cyclase (AC)activity. According to certain aspects, modulation of at least one ofcellular membrane potential and cellular membrane conductivity comprisesmodulation of intracellular signal transduction associated with at leastone condition or symptom selected from the group consisting of: dry eye,allergy irritation, corneal irritation, bacterial infection, andcellular damage.

Particular aspects provide for the invention to comprise administrationof the electrokinetically-altered aqueous fluid to a cell network orlayer, and further comprising modulation of an intercellular junctiontherein. In further aspects, the intracellular junction comprises atleast one selected from the group consisting of tight junctions, gapjunctions, zona adherins and desmasomes. In certain aspects, the cellnetwork or layers comprises at least one selected from the groupconsisting of corneal epithelial layer and corneal ‘endothelium’.

According to particular aspects, the electrokinetically-altered aqueousfluid is oxygenated, and wherein the oxygen in the fluid is present inan amount of at least 8 ppm, at least 15, ppm, at least 25 ppm, at least30 ppm, at least 40 ppm, at least 50 ppm, or at least 60 ppm oxygen atatmospheric pressure. In further aspects, the electrokinetically alteredaqueous fluid comprises at least one of a form of solvated electrons,and electrokinetically modified or charged oxygen species. In certainaspects, the form of solvated electrons or electrokinetically modifiedor charged oxygen species are present in an amount of at least 0.01 ppm,at least 0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, atleast 5 ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or atleast 20 ppm. According to additional aspects, theelectrokinetically-altered aqueous fluid comprises a form of solvatedelectrons stabilized, at least in part, by molecular oxygen.

In certain aspects, the ability to alter at least one of cellularmembrane potential and cellular membrane conductivity persists for atleast two, at least three, at least four, at least five, at least 6, atleast 12 months, or longer periods, in a closed gas-tight container. Infurther aspects, the electrokinetically-altered aqueous fluid comprisesa contact lens solution or multi-use contact lens solution. According toadditional aspects, the contact lens solution or multi-use contact lenssolution comprises a storage solution, a wetting solution, or a cleaningsolution. In certain aspects, the electrokinetically-altered aqueousfluid inhibits the growth of Pseudomonas bacteria.

In particular aspects, the irritation, infection or inflammatory eyecondition is the result of a wound. In further aspects, the wound isselected from the group consisting of: lacerations, abrasions, rupture,puncture wounds, chemical, thermal, or radiation-induced burns, cuts,scrapes, incisions, blisters, ulcers, and surgical wounds. In additionalaspects, the surgical wound is a result of at least one surgery selectedfrom the group consisting of: laser keratotomy, cataract removal, lensimplantation or removal, corneal alterations, laser-assisted in situkeratomileusis (LASIK), intraLASIK, extracapsular surgery,phacoemulsification, vitreoretinal surgery, glaucoma procedures,neuro-ophthalmic surgery, and strabismus surgery.

In further aspects, the irritation, infection or inflammatory eyecondition or disease comprises dry eye, and wherein at least one sign orsymptom selected from the group consisting of: decrease in aqueous tearproduction, increase in tear evaporation, and increase in cellularinflammation is ameliorated.

According to certain aspects, the ameliorating is assessed by at leastone measurement selected from the group consisting of corneal staining,conjunctival staining, tear volume, bulbar conjunctival erythema, tearfilm height, ocular surface healing, level of infection, level ofinflammation, level of ocular surface damage, and level of inferior tearmeniscus.

In particular aspects, the contacting comprises topically applying saidfluid to said eye. In further aspects, the treatment comprises anadjunct therapy and said subject further undergoes a primary treatmentfor an eye condition. According to further aspects, the primarytreatment comprises administering to said subject at least onemedication selected from the group consisting of artificial tears,anti-inflammatory medications, steroids, antihistamines, antibiotics,and vasoconstrictor medications. In additional aspects, theadministering comprises topically or orally administering saidmedication to the subject.

In further aspects, the contacting comprises topically or systemicallycontacting said eye of the subject. According to additional aspects, thetreatment results in a reduction in corneal or conjuctival staining. Incertain aspects, the treatment results in an increase in tear volume ortear film height.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial cross-section, partial block diagram of a prior artmixing device.

FIG. 2 is block diagram of an exemplary embodiment of a mixing device.

FIG. 3 is an illustration of an exemplary system for delivering a firstmaterial to the mixing device of FIG. 2.

FIG. 4 is a fragmentary partial cross-sectional view of a top portion ofthe mixing device of FIG. 2.

FIG. 5 is a fragmentary cross-sectional view of a first side portion ofthe mixing device of FIG. 2.

FIG. 6 is a fragmentary cross-sectional view of a second side portion ofthe mixing device of FIG. 2.

FIG. 7 is a fragmentary cross-sectional view of a side portion of themixing device of FIG. 2 located between the first side portion of FIG. 5and the second side portion of FIG. 6.

FIG. 8 is a perspective view of a rotor and a stator of the mixingdevice of FIG. 2.

FIG. 9 is a perspective view of an inside of a first chamber of themixing device of FIG. 2.

FIG. 10 is a fragmentary cross-sectional view of the inside of a firstchamber of the mixing device of FIG. 2 including an alternate embodimentof the pump 410.

FIG. 11 is a perspective view of an inside of a second chamber of themixing device of FIG. 2.

FIG. 12 is a fragmentary cross-sectional view of a side portion of analternate embodiment of the mixing device.

FIG. 13 is a perspective view of an alternate embodiment of a centralsection of the housing for use with an alternate embodiment of themixing device.

FIG. 14 is a fragmentary cross-sectional view of an alternate embodimentof a bearing housing for use with an alternate embodiment of the mixingdevice.

FIG. 15 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor approaches (but is not aligned with) anaperture of the stator.

FIG. 16 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen the through-hole of the rotor is aligned with the aperture of thestator.

FIG. 17 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor that was previously aligned with theaperture of the stator is no longer aligned therewith.

FIG. 18 is a side view of an alternate embodiment of a rotor.

FIG. 19 is an enlarged fragmentary cross-sectional view taken through aplane orthogonal to an axis of rotation of the rotor depicting analternate configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 20 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting a configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 21 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting an alternate offset configuration of through-holesformed in the rotor and through-holes formed in the stator.

FIG. 22 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 23 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 24 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 25 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 26 is an illustration of an electrical double layer (“EDL”) formednear a surface.

FIG. 27 is a perspective view of a model of the inside of the mixingchamber.

FIG. 28 is a cross-sectional view of the model of FIG. 27.

FIG. 29 is an illustration of an experimental setup.

FIG. 30 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored a 500 ml thin walledplastic bottle and a 1,000 ml glass bottle each capped at 650Fahrenheit.

FIG. 31 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored in a 500 ml plasticthin walled bottle and a 1,000 ml glass bottle both refrigerated at 39°Fahrenheit.

FIG. 32 illustrates the dissolved oxygen retention of a 500 ml beveragefluid processed with oxygen in the mixing device of FIG. 2.

FIG. 33 illustrates the dissolved oxygen retention of a 500 ml braunbalanced salt solution processed with oxygen in the mixing device ofFIG. 2.

FIG. 34 illustrates a further experiment wherein the mixing device ofFIG. 2 is used to sparge oxygen from water by processing the water withnitrogen in the mixing device of FIG. 2.

FIG. 35 illustrates the sparging of oxygen from water by the mixingdevice of FIG. 2 at standard temperature and pressure.

FIG. 36 is an illustration of an exemplary nanocage.

FIGS. 37A and B illustrate Rayleigh scattering effects of anoxygen-enriched fluid;

FIG. 38 illustrates the cytokine profile of a mitogenic assay in thepresence of a gas-enriched fluid and deionized control fluid; and

FIG. 39 illustrates the difference in the growth rates of Pseudomonasbacteria at various dissolved oxygen saturation ratios.

FIGS. 40A and 40B illustrate in vitro healing of wounds using anoxygen-enriched cell culture media and a non-gas-enriched media.

FIGS. 41A through 41F show histological cross-sections of dermal andepidermal in vivo wound healing.

FIG. 42 illustrates the expression of Hale's stain in treated andcontrol healing wounds, used to detect acid mucopolysaccharides, such ashyaluronic acid;

FIG. 43 illustrates the expression of von Willebrand's Factor stain usedto detect angiogenesis in treated and control healing wounds;

FIG. 44 illustrates the detection of Luna's stain used to detect elastinin treated and control healing wounds;

FIG. 45 illustrates the number of mast cells per visual field fortreated and control healing wounds;

FIG. 46 illustrates the percentage of dead cells at separate time pointsin a corneal fibroblast assay using inventive gas-enriched culture mediaand control culture media,

FIG. 47 illustrates the shelf life of the inventive gas-enriched fluidin a polymer pouch;

FIG. 48 illustrates the results of contacting splenocytes with MOG inthe presence of pressurized pot oxygenated fluid (1), inventivegas-enriched fluid (2), or control deionized fluid (3).

FIGS. 49-58 show the results of whole blood sample evaluations ofcytokines.

FIGS. 59-68 show the corresponding cytokine results of bronchoalveolarlavage fluid (BAL) sample evaluations.

FIGS. 69-75 shows studies where the Bradykinin B2 membrane receptor wasimmobilized onto aminopropylsilane (APS) biosensor. The Sample plate setup was as designated in FIG. 69 and the binding of Bradykinin to theimmobilized receptor was assessed according to the sample set up asdesignated in FIG. 71. Results of Bradykinin binding are shown in FIG.72. Bradykinin binding to the receptor was further titrated according tothe set-up as designated in FIG. 73. As indicated in FIG. 74, Bradykininbinding to the B2 receptor was concentration dependent, and bindingaffinity was increased in the proprietary gas-enriched saline fluid ofthe instant disclosure compared to normal saline. Stabilization ofBradykinin binding to the B2 receptor is shown in FIG. 75.

FIGS. 76-83 show data showing the ability of particular embodimentsdisclosed herein to affect regulatory T cells. The study involvedirradiating antigen presenting cells, and introducing antigen and Tcells.

FIG. 84 shows that the inventive electrokinetically generated fluidsdecreased serum uptake of salmon calcitonin and an animal model. Theresults are consistent with enhancement of tight junctions.

FIGS. 85-89 show the expression levels of of tight junction-relatedproteins in lung tissue from the animal model used to generate the dataof FIG. 84.

FIGS. 90-94 show data obtained from human foreskin keratinocytes exposedto RDC1676-01 (sterile saline processed through the instant proprietarydevice with additional oxygen added; gas-enriched electrokineticallygenerated fluid (Rev) of the instant disclosure) showing up-regulationof NOS1 and 3, and Nostrin, NOS3.

FIGS. 95 and 96 show data supporting localized electrokinetic effects(voltage/current) occurring in a mixing device comprising insulatedrotor and stator features to allow for detection of voltage/currenteffects during electrokinetic fluid generation.

FIGS. 97A-C show results of nuclear magnetic resonance (NMR) studiesconducted to further characterize the fundamental nature of theinventive electrokinetically generated fluids. The electrokineticallygenerated fluids increased the ¹³C-NMR line-widths of the reporterTrehalose solute.

FIGS. 98 and 99 show results of voltametric studies (i.e., square wavevoltametry (FIG. 98) and stripping polarography (FIG. 99)) conducted tofurther characterize the fundamental nature of the inventiveelectrokinetically generated fluids. Square wave voltametry peakdifferences (with respect to control) unique to the electrokineticallygenerated fluids were observed at −0.14V, −0.47V, −1.02V and −1.36V.Pronounced polaragraphic peaks were seen at −0.9 volts for theelectrokinetically generated Revera and Solas fluids, and the spectra ofthe non-electrokinetically generated blank and saline control fluidsshow characteristic peaks at −0.19 and −0.3 volts that are absent in thespectra for the electrokinetically generated fluids.

FIGS. 100-106 show results of patch clamping techniques that assessedthe effects of the electrokinetically generated fluid test on epithelialcell membrane polarity and ion channel activity. The results indicatethat the inventive electrokinetically generated fluids affect avoltage-dependent contribution of the whole-cell conductance.

FIGS. 107A-D and 108A-D show data indicating that the inventiveelectrokinetically generated fluids (e.g., RDC1676-00, RDC1676-01,RDC1676-02 and RDC1676-03) protected against methacholine-inducedbronchoconstriction when administered alone or as diluents for albuterolsulfate in male guinea pigs.

FIGS. 109-114 show results of budesonide experiments performed to assessthe airway anti-inflammatory properties of the inventiveelectrokinetically generated fluids in a Brown Norway rat ovalbuminsensitization model. The inventive electrokinetically generated fluidsdecreased eosinophil count, showed strong synergy with Budesonide indecreasing eosinophil count, decreased Penh values, increased TidalVolume, decreased blood levels of Eotaxin, significantly enhanced theBlood levels of two major key anti-inflammatory cytokines, IL10 andInterferron gamma at 6 hours after challenge as a result of treatmentwith the inventive electrokinetically generated fluid (e.g., Rev 60)alone or in combination with Budesonide, and decreased systemic levelsof Rantes. The data show that there is a substantial synergistic effectof Budesonide 750 ug/kg and the inventive electrokinetically generatedfluids (e.g., Rev 60).

FIG. 115 shows that the inventive electrokinetically generated fluid(e.g., Revera 60 and Solas) reduced DEP-induced TSLP receptor expressionin bronchial epithelial cells (BEC) by approximately 90% and 50%,respectively, whereas whereas normal saline (NS) had only a a marginaleffect.

FIG. 116 shows the inventive electrokinetically generated fluid (e.g.,Revera 60 and Solas) inhibited the DEP-induced cell surface bound MMP9levels in bronchial epithelial cells by approximately 80%, and 70%,respectively, whereas normal saline (NS) had only a marginal effect.

FIGS. 117A-C demonstrate the results of a series of patch clampingexperiments that assessed the effects of the electrokineticallygenerated fluid (e.g., RNS-60 and Solas) on epithelial cell membranepolarity and ion channel activity at two time-points (15 min (leftpanels) and 2 hours (right panels)) and at different voltage protocols.

FIGS. 118 A-C show, in relation to the experiments relating to FIGS. 117A-C, the graphs resulting from the subtraction of the Solas current datafrom the RNS-60 current data at three voltage protocols (A. steppingfrom zero mV; B. stepping from −60 mV; C. stepping from −120 mV) and thetwo time-points (15 mins (open circles) and 2 hours (closed circles)).

FIGS. 119 A-D demonstrate the results of a series of patch clampingexperiments that assessed the effects of the electrokineticallygenerated fluid (e.g., Solas (panels A. and B.) and RNS-60 (panels C.and D.)) on epithelial cell membrane polarity and ion channel activityusing different external salt solutions and at different voltageprotocols (panels A. and C. show stepping from zero mV; panels B. and D.show stepping from −120 mV).

FIGS. 120 A-D show, in relation to the experiments relating to FIGS. 119A-D, the graphs resulting from the subtraction of the CsCl current data(shown in FIG. 119) from the 20 mM CaCl₂ (diamonds) and 40 mM CaCl₂(filled squares) current data at two voltage protocols (panels A. and C.stepping from zero mV; B. and D. stepping from −120 mV) for Solas(panels A. and B.) and Revera 60 (panels C. and D.).

FIG. 121 A shows 1 mm2 AFM scan for RNS60-1 (rns60-1 1 um 3D.jpg). Thesmall peaks (“1”) represent hydrophobic nanobubbles which are ˜20 nmwide and ˜1.5 nm tall or smaller.

FIG. 121 B shows 1 mm2 scan for PNS60-1 (pp60-1 1 um 3d.jpg). This scanreveals peaks (“2”) (hydrophobic nanobubbles) that are substantiallylarger (˜60 nm wide and ˜5 nm tall) than those visible with RNS60-1.

DETAILED DESCRIPTION

The surface of the animal eye, particularly the human eye, is normallybathed by a tear film that is secreted by tiny glands around the eye.The tear film is primarily composed of three layers: mucous, water, andoil. The mucous layer is closest to the eye organ and serves as ananchor for the tear film to adhere to the eye. The middle layer is anaqueous layer, and the outer oil layer seals the tear film and preventsevaporation. The tear film also contains various nutritive andprotective proteins and peptides. The tear film serves several purposes:it keeps the eye moist, creates a smooth surface for light to passthrough the eye, nourishes the front of the eye, and provides protectionfrom injury and infection.

The eye, particularly the cornea, can become dry, irritated, or damagedby trauma, disease, natural aging, environmental factors (such aspollutants and allergens), or by wearing contact lenses. In some cases,individuals may experience burning, a feeling of dryness, scratchiness,itchiness, or persistent irritation or inflammation. This irritation canlead to infection and/or visual impairment if not treated properly.

As summarized above, the most common approach to treatment of irritatedand/or dry eyes has been to supplement the natural ocular tear filmusing so-called artificial tears instilled throughout the day. Otherapproaches include the use of lubricants, and ocular inserts thatprovide a tear substitute or stimulation of endogenous tear production.Novel, safe and effective approaches are needed, and are providedherein.

Certain embodiments disclosed herein relate to providing compositionsand methods of treatment of at least one symptom or sign of an eye (e.g.corneal) condition or disorder by contacting the site or administeringto a subject, a therapeutic and/or cosmetic composition comprising agas-enriched fluid. In certain specific embodiments, the gas-enrichedfluid comprises oxygen-enriched water.

Certain embodiments disclosed herein provide compositions and methodsfor eye care and treatment comprising administering a therapeuticcomposition comprising at least one electrokinetically generated fluid(including gas-enriched (e.g., oxygen enriched) electrokineticallygenerated fluids) as disclosed herein for regulating or modulatingintracellular signal transduction by modulation of at least one ofcellular membranes, membrane potential, and membrane proteins such asmembrane receptors, including but not limited to G protein coupledreceptors. Particular aspects relate to modulating (e.g., treating orpreventing) at least one disease or condition or symptom thereofassociated with eye care and treatment.

According to particular aspects, the introduction of oxygen-rich salinein therapeutic applications has beneficial effects on human tissues invivo. Applicants' microarray analyses have confirmed theanti-inflammatory nature of the inventive fluids through an increase inthe level of anti-inflammatory cytokines, and such fluids wouldtherefore be advantageous in dry eye as well as otherinflammatory-mediated conditions. According to additional aspects, theapplication of oxygen-rich saline to cultured cells shows no significantlevels of toxicity that would preclude in vivo use.

Certain embodiments disclosed herein relate to providing compositionsand methods of treatment of at least one symptom or sign of an eye (e.g.corneal) condition or disorder by contacting the site or administeringto a subject, a therapeutic and/or cosmetic composition comprising anelectrokinetically generated fluid (e.g., gas-enriched fluid) asdescribed herein. In certain specific embodiments, a gas-enriched fluidcomprises oxygen-enriched water.

Particular embodiments disclosed herein relate to compositions andmethods for treating non-responding dry eye patients with pre-existingocular surface damage when added to their current treatment regime.Certain other embodiments disclosed herein relate to topically appliedcompositions and methods for treating dry eyes as a safe and effectiveadjunct therapy in conjunction with concomitant therapies. Still otherembodiments relate to compositions and methods for beneficially treatingsevere dry eye disease that is unresponsive to standard therapies byreducing at least one sign or symptom of severe dry eye disease.

Other particular embodiments relate to compositions and methods fortreating allergy eye patients. In certain embodiments, the compositionsdescribed herein comprise a preservative free, buffered saline eyerewetting drop. In particular embodiments, the rewetting drop is furthergas-enriched. In specific embodiments the gas-enriched fluid isoxygen-enriched water or oxygen-enriched saline.

Electrokinetically-Generated Fluids:

“Electrokinetically generated fluid,” as used herein, refers toApplicants' inventive electrokinetically-generated fluids generated, forpurposes of the working Examples herein, by the exemplary Mixing Devicedescribed in detail herein (see also US200802190088 and WO2008/052143,both incorporated herein by reference in their entirety). Theelectrokinetic fluids, as demonstrated by the data disclosed andpresented herein, represent novel and fundamentally distinct fluidsrelative to prior art non-electrokinetic fluids, including relative toprior art oxygenated non-electrokinetic fluids (e.g., pressure potoxygenated fluids and the like). As disclosed in various aspects herein,the electrokinetically-generated fluids have unique and novel physicaland biological properties including, but not limited to the following:

In particular aspects, the electrokinetically altered aqueous fluidcomprise an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide, upon contact ofa living cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity.

In particular aspects, electrokinetically-generated fluids refers tofluids generated in the presence of hydrodynamically-induced, localized(e.g., non-uniform with respect to the overall fluid volume)electrokinetic effects (e.g., voltage/current pulses), such as devicefeature-localized effects as described herein. In particular aspectssaid hydrodynamically-induced, localized electrokinetic effects are incombination with surface-related double layer and/or streaming currenteffects as disclosed and discussed herein.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to modulate ¹³C-NMR line-widths of reporter solutes (e.g.,Trehelose) dissolved therein. NMR line-width effects are in indirectmethod of measuring, for example, solute ‘tumbling’ in a test fluid asdescribed herein in particular working Examples.

In particular aspects, the electrokinetically altered aqueous fluids arecharacterized by at least one of: distinctive square wave voltametrypeak differences at any one of −0.14V, −0.47V, −1.02V and −1.36V;polarographic peaks at −0.9 volts; and an absence of polarographic peaksat −0.19 and −0.3 volts, which are unique to the electrokineticallygenerated fluids as disclosed herein in particular working Examples.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to alter cellular membrane conductivity (e.g., avoltage-dependent contribution of the whole-cell conductance as measurein patch clamp studies disclosed herein).

In particular aspects, the electrokinetically altered aqueous fluids areoxygenated, wherein the oxygen in the fluid is present in an amount ofat least 15, ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, atleast 50 ppm, or at least 60 ppm dissolved oxygen at atmosphericpressure. In particular aspects, the electrokinetically altered aqueousfluids have less than 15 ppm, less that 10 ppm of dissolved oxygen atatmospheric pressure, or approximately ambient oxygen levels.

In particular aspects, the electrokinetically altered aqueous fluids areoxygenated, wherein the oxygen in the fluid is present in an amountbetween approximately 8 ppm and approximately 15 ppm, and in this caseis sometimes referred to herein as “Solas.”

In particular aspects, the electrokinetically altered aqueous fluidcomprises at least one of solvated electrons (e.g., stabilized bymolecular oxygen), and electrokinetically modified and/or charged oxygenspecies, and wherein in certain embodiments the solvated electronsand/or electrokinetically modified or charged oxygen species are presentin an amount of at least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm,at least 1 ppm, at least 3 ppm, at least 5 ppm, at least 7 ppm, at least10 ppm, at least 15 ppm, or at least 20 ppm.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to alter cellular membrane structure or function (e.g.,altering of a conformation, ligand binding activity, or a catalyticactivity of a membrane associated protein) sufficient to provide formodulation of intracellular signal transduction, wherein in particularaspects, the membrane associated protein comprises at least one selectedfrom the group consisting of receptors, transmembrane receptors (e.g.,G-Protein Coupled Receptor (GPCR), TSLP receptor, beta 2 adrenergicreceptor, bradykinin receptor, etc.), ion channel proteins,intracellular attachment proteins, cellular adhesion proteins, andintegrins. In certain aspects, the effected G-Protein Coupled Receptor(GPCR) interacts with a G protein α subunit (e.g., Gα_(s), Gα_(i),Gα_(q), and Gα₁₂).

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to modulate intracellular signal transduction, comprisingmodulation of a calcium dependant cellular messaging pathway or system(e.g., modulation of phospholipase C activity, or modulation ofadenylate cyclase (AC) activity).

In particular aspects, the electrokinetically altered aqueous fluids arecharacterized by various biological activities (e.g., regulation ofcytokines, receptors, enzymes and other proteins and intracellularsignaling pathways) described in the working Examples and elsewhereherein.

In particular aspects, the electrokinetically altered aqueous fluidsdisplay synergy with metformin as shown in working Examples herein

In particular aspects, the electrokinetically altered aqueous fluidsreduce DEP-induced TSLP receptor expression in bronchial epithelialcells (BEC) as shown in working Examples herein.

In particular aspects, the electrokinetically altered aqueous fluidsinhibit the DEP-induced cell surface-bound MMP9 levels in bronchialepithelial cells (BEC) as shown in working Examples herein.

In particular aspects, the biological effects of the electrokineticallyaltered aqueous fluids are inhibited by diphtheria toxin, indicatingthat beta blockade, GPCR blockade and Ca channel blockade affects theactivity of the electrokinetically altered aqueous fluids (e.g., onregulatory T cell function) as shown in working Examples herein.

In particular aspects, the physical and biological effects (e.g., theability to alter cellular membrane structure or function sufficient toprovide for modulation of intracellular signal transduction) of theelectrokinetically altered aqueous fluids persists for at least two, atleast three, at least four, at least five, at least 6 months, or longerperiods, in a closed container (e.g., closed gas-tight container).

Anti-Inflammatory Activity of the Electrokinetically-GeneratedGas-Enriched Fluids and Solutions:

According to certain aspects of the present invention, the gas-enrichedfluids and/or solutions disclosed herein have anti-inflammatoryproperties and effects, and can be used as anti-inflammatory agents forthe treatment of subjects afflicted by diseases or disorders relating toinflammation. FIG. 38 shows the experimental results of cytokineprofiles in stimulated lymphocytes from a healthy blood donor. As can beseen in FIG. 38, the inventive oxygen-enriched fluid (water) affected adown regulation of particular cytokines, especially IL-6, IL-8, andIL-1β.

Increased production of pro-inflammatory cytokines has been implicatedin the pathogenesis of numerous inflammatory and autoimmune diseases.Secretion of TNFα is a primary event in the initiation of theinflammatory cascade (Brennan F. M., et. al. Lancet, 1989, 2:244-7;Haworth C, et. al. Eur. J. Immunol. 1991, 21:2575-2579) and directlycontributes to the initiation and maintenance of inflammatory andautoimmune diseases. Other pro-inflammatory cytokines also play a role,including interleukin 1β (IL-1β), IL-6, IL-8, IL-12 nitric oxide, IFN-γand GM-CSF, while anti-inflammatory cytokines such as IL-10 may reducedisease. Cells of the immune system, macrophages in particular, secretemany of these cytokines in response to activating stimuli.

A variety of cell types are involved in the inflammatory process.Overproduction of TNFα by monocytes, macrophages and other immune cellsis a key element in the pathogenesis of a multitude of diseases.Macrophages and T-cells in particular play a central role in theinitiation and maintenance of the immune response. Once activated bypathological or immunogenic stimuli, macrophages respond by releasing ahost of cytokines, including TNF-α, IL-1β, IL-8, IL-12, nitric oxide(NO), IL-6, GM-CSF, G-CSF, M-CSF and others. T-cells release IL-2, IL-4,INF-γ, and other inflammatory cytokines. These cytokines activate otherimmune cells and some can also act as independent cytotoxic agents.Excessive release of macrophage and T-cell derived inflammatorymediators can particularly lead to damage of normal cells andsurrounding tissues.

Pro-inflammatory cytokines have been implicated to contribute to manydiseases and disease symptoms including, diabetes. The induction of NOfrom smooth muscle cells mediates decreased mean arterial pressure andsystemic vascular resistance during septic shock, suggesting afundamental role for NO. Thus, therapies that target downregulatoryeffects on IL-8, IL-1β, and NO could be beneficial in the treatment ofinflammatory diseases or disorders, including sepsis, septic shock,endotoxic shock and diabetes.

Overproduction of TNFα contributes to the clinical features of numerousautoimmune diseases such as diabetes and rheumatoid arthritis. Systemiclupus erythematosus (SLE) is also precipitated by increased IL-1β andTNFα levels. Within lupus patients, serum C-reactive protein, IL-1β andTNFα levels were higher than in controls, suggesting that an increasedinflammatory response plays a role in the disease (Liou L. B. Clin. Exp.Rheumatol. 2001, 19:515-523). A study of patients with one form of SLE,neuropsychiatric lupus erythematosus (NPLE), showed that the number ofperipheral blood mononuclear cells expressing mRNA for TNFα as well asthe cerebrospinal fluid level of NO metabolites correlated with NPLEdisease severity (Svenungsson E., et al. Ann. Rheum. Dis. 2001,60:372-9).

IL-1 and TNFα play a central role in various acute as well as chronicresponses in animal models. Additionally, IL-11, IFNα and IFNβ may alsoup-regulate inflammatory reactions. Conversely, several cytokines may beinvolved in down-regulation of inflammatory responses (i.e. IL-4, IL-10,IL-13, among others). As set forth in Example 1, cells contacted withthe inventive gas-enriched fluid showed an increase in IFN-γ levels withT3 antigen than in the control culture media with T3 antigen, while IL-8was lower in the inventive gas-enriched culture media with T3 antigenthan in the control culture media with T3 antigen. Additionally, IL-6,IL-8, and TNF-α levels were lower in the inventive gas-enriched mediawith PHA, than in the control media with PHA, while IL-1β levels werelower in the inventive gas-enriched fluid with PHA when compared withcontrol media with PHA. In the inventive gas-enriched media alone, IFN-γlevels were higher than in control media. These results are consistentwith an anti-inflammatory microenvironment.

NO is recognized as a mediator and regulator of inflammatory responses.It possesses cytotoxic properties toward pathogens, but can also havedeleterious effects on the subject's own tissues. (Korhonen et al., CurrDrug Targets Inflamm Allergy 4(4): 471-9, 2005). NO reacts with solubleguanylate cyclase to form cyclic guanosine monophosphate (cGMP), whichmediates many of the effects of NO. NO can also interact with molecularoxygen and superoxide anion to produce reactive oxygen species that canmodify various cellular functions. These indirect effects of NO have asignificant role in inflammation, where NO is produce in high amounts byinducible NO synthase (iNOS) and reactive oxygen species are synthesizedby activated inflammatory cells. In fact, overproduction of NOcontributes to impairment of both muscle cell insulin action and β cellfunction in obesity, which can lead to insulin resistance and diabetes.

NO can be produced by keratinocytes, fibroblasts, endothelial cells, andpossibly others. Some of the vascular actions of NO includevasodilation, inhibiting platelet adhesion to the vascular endothelium,inhibiting leukocyte adhesion to the vascular endothelium, andscavenging superoxides. (Shah et al., Env. Health Persp. v. 106 (5):1139-1143.)

Furthermore, inhibition of NO synthesis has been shown to delay woundcontraction, alter collagen organization, and alter neoepidermisthickness. (Amadeu and Costa, J. Cutan. Pathol 33: 465-473, 2006.) Mastcell migration and angiogenesis in wounds is also affected by inhibitionof NO. (Id.) Without being bound to any particular theory of mechanism,in certain embodiments the inventive gas-enriched fluids may bemodulating localized and/or cellular NO production, or degradation,consistent with the spectrum of wound healing effects illustrated in theExamples section disclosed herein. Due to variable pathways ofregulation, in certain embodiments, the inventive gas-enriched fluid mayincrease NO production and/or retard NO degradation, whereas in othercertain embodiments, the inventive gas-enriched fluid may decrease NOproduction and/or hasten NO degradation.

Specifically, wounds treated with oxygen-enriched saline solution showedan increase in wound healing at days 4 through 11, and between days 3and 11, the new epidermis in wounds treated with the oxygen-enrichedsaline solution migrated at two to four times as fast as the epidermisof the wounds treated with the normal saline solution, as set forth inExample 9 herein. The study also showed that between 15 and 22 days,wounds treated by the oxygen-enriched saline solution differentiated ata more rapid rate as evidenced by the earlier formation of more matureepidermal layers. At all stages, the thickening that occurs in theepidermis associated with normal healing did not occur within the woundstreated by the oxygen-enriched saline solution.

Thus, in accordance with this spectrum of wound healing effects, butwithout wishing to be bound by any particular theory, it is believedthat the oxygen-enriched saline solution may modulate the localizedand/or cellular level of NO within the wounds. NO modulates growthfactors, collagen deposition, inflammation, mast cell migration,epidermal thickening, and neovascularization in wound healing.Furthermore, nitric oxide is produced by an inducible enzyme that isregulated by oxygen.

In the case of mast cell migration, differences also occurred in earlyand late migration for the oxygen-enriched solution. This is consistentwith what is known in the art regarding inhibition of NO synthesis(Amadeu and Costa, J. Cutan Pathol 33: 465-473, 2006).

Referring now to FIG. 41A through 41F, various illustrations compare thewound healing results of the porcine epidermal tissues with or withoutoxygen-enriched saline solution. As can be seen, the healing of thecontrol wound and of the wound using the oxygen-enriched saline solutionwas followed for days 1, 4 and 16.

FIG. 41A illustrates the wound healing for the control wound on day 1.As can be seen, the wound shows epidermal/dermal thickening and a lossof contour. FIG. 41B illustrates the wound healing on day 1 for thewound treated using the oxygen-enriched saline solution. The wound showsnormal epidermal/dermal thickness and normal contouring is typical on anew wound.

Referring now to FIGS. 41C and 41D, there are illustrated the woundhealing for the control wound on day 4 and the wound healing for thewound treated with the oxygen-enriched saline solution on day 4. For thecontrol wound illustrated in FIG. 41C, the wound shows a 600 micronepidermal spur. In the wound treated with the oxygen-enriched salinesolution in FIG. 41D, there is illustrated a 1200 micron epidermal spur.Thus, in the first 4 days of the experiment, the epidermal spur createdin the wound treated using the oxygen-enriched saline solution shows anepidermal growth rate of twice of that of the wound that was not treatedwith the oxygen-enriched saline solution.

Referring now to FIG. 41E, there is illustrated the control wound at day16. The wound shows less differentiated epidermis with loss ofepidermal/dermal contour than that illustrated by the wound treated withthe oxygen-enriched saline solution illustrated in FIG. 41F. FIG. 41Fshows more differentiated epidermis and more normal epidermal/dermalcontouring in the wound.

In the first two phases of the inflammatory process, the foreign body iseither destroyed, for example, if the foreign body is an organism, orthe tissue around it is loosened, for example, if it is a splinter. Inthe healing phase, the inflammation begins to subside; individual bloodvessels and vascular patterns become normal once again; and repair ofthe wound commences. The three main events in the repair process are (1)formation of new connective tissue by proliferating fibroblasts; (2)regeneration of epithelium; and (3) outgrowth of new capillaries.

Even before the inflammation subsides, fibroblasts begin moving into theinjured area from the surrounding normal tissue, where they usuallyexist in a dormant state. They migrate by an amoeboid movement alongstrands of fibrin and distribute themselves throughout the healing area.Once fixed into position in the injured tissue, they begin to synthesizecollagen and secrete this protein, which arranges itself into fibers.The fibers orient themselves with their longitudinal axes in thedirection of the greatest stress. As the collagen bundles grow infirmness, the fibroblasts gradually degenerate and attach closely to thebundles, and the injured area transforms into scar tissue.

Simultaneously with scar tissue formation, the intact epidermal cells onthe edge of the wound begin to proliferate and move, as one sheet,toward the center of the injured area. As the inflammation subsides, aneed for a direct supply of blood arises, and angiogenesis occurs at thewound site.

Inflammation is a complex process that involves multiple cell types. Forexample, mast cells release mediators that trigger an early phase ofvasodilation, accompanied by the separation of endothelial cells andexposure of collagen fibers in the subendothelial layer. Fibers in theintercellular gaps that form in blood vessels trap platelets and triggerthe release of mediators from these cells.

In addition to platelets, the exposed collagen fibers also interact withproteins of the plasma that filter through the pores of the dilatedvessel wall, including the triggering factor of the blood-clottingcascade, increased vasodilation, increased blood vessel permeability,and chemotaxis.

Additionally, the complement cascade can be activated by severalstimuli: the injured blood vessels, the proteolytic enzymes released bythe damaged cells, the membrane components of any participatingbacteria, and antigen-antibody complexes. Some of the activatedcomplement components act as chemotactic factors, responsible for theinflux of leukocytes into the inflamed area, while others facilitatephagocytosis and participate in cell lysis.

In particular aspects, inventive gas-enriched fluids or solutions alsoregulate at least one cytokine involved in at least one aspect ofinflammation, the cytokine(s) including, but not limited to MAF(macrophage activating factor), MMIF (macrophage migration inhibitionfactor), MCF (macrophage chemotactic factor), LMIF (leukocyte migrationinhibition factor), HRFs (histamine releasing factors), TF (transferfactors), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, etc.), TNF-α, TNF-β,interferons (IFN-α, IFN-β, IFN-γ, IFN-ζ, IFN-δ, etc.), G-CSF(granulocyte colony stimulating factor), GM-CSF (granulocyte-macrophageCSF), M-CSF (macrophage CSF), multi-CSF (IL-3), fibroblast growth factor(aFGF, bFGF), EGF (epidermal growth factor), NGF (nerve growth factor),PDGF (platelet-derived growth factor), VEGF (vascular endothelial growthfactor), transforming growth factors (TGF-α, TGF-β, etc.), NAP-2(neutrophil-activating protein 2), PF-4 (platelet factor 4),thromboglobulin, MCP-1 (monocyte chemoattractant protein 1), MCP-3,MIP-1α, MIP-1β−+ (macrophage inflammatory proteins), RANTES (regulatedupon activation normal T expressed and presumably secreted chemokine),HSPs (heat shock proteins), GRPs (glucose-regulated proteins),ubiquitin, and others.

Thus, in certain embodiments, the gas-enriched fluids and/or therapeuticcompositions increase production and/or secretion of anti-inflammatorymolecules or cytokines or decrease the degradation of anti-inflammatorymolecules or cytokines, thereby alleviating or preventing at least onesymptom of inflammation. In other embodiments, the gas-enriched fluidsand/or therapeutic compositions of the present invention may decreaseproduction and/or secretion of pro-inflammatory molecules or cytokinesor increase the degradation of pro-inflammatory molecules or cytokines,thereby alleviating or preventing at least one symptom of inflammation.

Previous studies had shown a critical role of anti-MOG antibodies inaugmentation of demyelination and worsening of EAE (experimentalautoimmune encephalomyelitis), an animal model system for the humanautoimmune disorder of rheumatoid arthritis (Linington, et al. 1992. J.Neuroimmunol. 40:219-224). Additionally, antibodies against MOG havebeen implicated in the pathogenesis of multiple sclerosis. (Berger etal. N. Engl. J. Med. Jul. 10, 2003; 349(2):139-45).

As set forth in FIG. 48 and Example 12, the inventive gas-enriched fluidof the present invention amplifies the lymphocyte response to an antigenfor which an animal was previously primed. As indicated in FIG. 48,lymphocyte proliferation was greater for response to MOG challenge whencultured in fluid reconstituted with the inventive gas-enriched fluidcomprising solvated electrons, when compared with pressurized,oxygenated fluid (pressure pot) or control deionized fluid.

Inventive Gas-Enriched Fluids and Solutions

Diffusing or enriching a fluid with another fluid may result in asolution or suspension of the two fluids. In particular, enriching aliquid with a gas (e.g. oxygen) may be beneficial for certainapplications, including therapeutic treatments. As utilized herein,“fluid,” may generally refer to a liquid, a gas, a vapor, a mixture ofliquids and/or gases, or any combination thereof, for any particulardisclosed embodiment. Furthermore, in certain embodiments a “liquid” maygenerally refer to a pure liquid or may refer to a gel, sol, emulsion,fluid, colloid, dispersion, or mixture, as well as any combinationthereof; any of which may vary in viscosity.

In particular embodiments, the dissolved gas comprises oxygen. In otherparticular embodiments, the dissolved gas comprises nitric oxide. Instill other embodiments, the dissolved gas comprises ambient air.

There are several art-recognized methods of gas-enriching fluids (suchas oxygen-enriching water). For example, a turbine aeration system canrelease air near a set of rotating blades of an impeller, which mixesthe air or oxygen with the water, or water can be sprayed into the airto increase its oxygen content. Additionally, other systems on themarket inject air or oxygen into the water and subject the water/gas toa large scale vortex. Naturally occurring levels of oxygen in water aretypically no more than 10 ppm (parts per million), which is consideredto be a level of 10 ppm dissolved oxygen. Tests on certain devices haveshown that under ideal conditions, the device can attain upwards ofapproximately 20 ppm dissolved oxygen, or twice the natural oxygenlevels of water. However, the water loses that high level of dissolvedoxygen very rapidly, and within minutes the water returns to having thebaseline of about 10 ppm dissolved oxygen.

In certain embodiments disclosed herein, a gas-enriched fluid of thepresent invention provides a cosmetic and/or therapeutic eye carebenefit. Certain embodiments disclosed herein relate to a cosmeticand/or therapeutic composition comprising a gas-enriched fluid of thepresent invention, and optionally at least one additional therapeuticagent, such as a pharmaceutical drug, a metal, a peptide, a polypeptide,a protein, a nucleotide, a carbohydrate or glycosylated protein, a fat(including oils or waxes), or other agent that prevents or alleviates atleast one symptom of a condition or disease associated with eyeirritation.

Compositions and methods are disclosed for treating the eye, or otherorgans and/or tissues in need thereof by topically applying an effectiveamount of a composition comprising a gas-enriched fluid.

As used herein, “treat,” “treating,” “treatment,” and any and allderivations thereof refer to using the compositions of the presentinvention either prophylactically to prevent signs of an eye conditionor disease, or cosmetically or therapeutically to ameliorate an existingcondition or disease. The term “treating” refers to, and includes,reversing, alleviating, inhibiting the progress of, or preventing adisease, disorder or condition, or one or more symptoms thereof; and“treatment” and “therapeutically” refer to the act of treating, asdefined herein.

A “therapeutically effective amount” is any amount of any of thecompounds utilized in the course of practicing the invention providedherein that is sufficient to reverse, alleviate, inhibit the progressof, or prevent a disease, disorder or condition, or one or more symptomsthereof. In one particular embodiment, the gas-enriched fluid of thepresent invention inhibits microbial growth. In another particularembodiment, the gas-enriched fluid of the present invention promotesapoptosis.

Microbial infections, particularly of Staphlycoccus, Streptococcus,yeast, Serratia, E. coli, Pseudomonas aeruginosa, and other microbialinfections, can cause devastating infections in the eyes if leftuntreated. Thus, in certain embodiments, the gas-enriched fluidcompositions and/or methods of the present invention includeanti-microbial agents, such as antifungal, antibiotic, or otheranti-microbial agents. Some examples of anti-microbial agents that maybe utilized with the gas-enriched fluid compositions and/or methodsinclude, but are not limited to, amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, paromomycin,geldanamycin, herimycin, loracarbef, ertapenem, imipenem/cilastatin,meropenem, cefadroxil, cefazolin, cefalotin/cefalothin, cephalexin,cefaclor, cefamandole, cefoxitin, cefuroxime, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefeprime, teicoplanin,vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spectinomycin, aztreonam,amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin,peperacillin, ticarcillin, bacitracin, colistin, polymyxin B,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, protosil,sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine,sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,arsphenamine, chloramphenicol, clindamycin, lincoamycin, ethambutol,fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid,metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide,quinupristin/dalfopristin, rifampin/rifampicin, tinidazole, miconazole,ketoconazole, clotrimazole, econazole, bifonazole, butoconazole,fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole,tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole,posaconazole, voriconazole, teronazole, terbinafine, amorolfine,naftifine, butenafine, anidulafungin, caspofungin, micafungin,ciclopirox, flucytosine, griseofulvin, Gentian violet, haloprogin,tolnaftate, undecylenic acid, and others.

The gas-enriched fluid compositions and/or methods provided herein mayinclude any form suitable for topical application, including a gas,aqueous or oil-based liquid, cream, lotion, balm, oil-and fluid mixture,gel, sol, emulsion, microemulsion, solution, suspension, or may beencapsulated in a liposome, microsponge, polymer matrix, or otherencapsulation technology adapted to aid in the delivery of thegas-enriched fluid to the areas of the eye in need thereof, or toenhance the stability of the composition or effectiveness of the methodemployed. In yet other embodiments, the gas-enriched fluid compositionis formulated for delivery by an instrument, such as ultrasound, to theaffected areas of the eye. In still other embodiments, the compositionfurther comprises an inert and physiologically-acceptable carrier ordiluent, enzymes, anti-microbial agents (anti-bacterial agents,anti-fungal agents, etc.), vasoconstrictors (such as epinephrine,naphazoline hydrochloride, tetrahydrozoline, etc.), acids (such as boricacid, hydrochloric acid, etc.), bases (such as sodium hydroxide, etc.),salts (such as sodium, potassium, calcium, etc.), polymers, alcohols(such as polyvinyl alcohol), cellulose or starch, dextrose, mannose,sucrose, or other carbohydrates; glycoproteins, proteins, polypeptidesor peptides, colors, fragrances, preservatives (such as edentatedisodium, chlorhexidine gluconate, etc.), or a mixture thereof. In otherrelated embodiments, the composition further comprises an activepharmaceutical drug or therapeutic drug substance or an active cosmeticsubstance. In one particular embodiment, the pharmaceutical ortherapeutic drug comprises an antihistamine, such as pheniraminemaleate.

In addition to the other compositions described herein, the presentdisclosure further contemplates combination therapies comprising atleast one embodiment of an inventive fluid in combination with atherapeutic agent, including but not limited to: ciclosporin, hyaluronicacid, carmellose, macrogol(s), dextran and hyprolose, sodium andcalcium, sodium and povidone, hypromellose, carbomer and others. Someparticular compositions containing these agents include, but are notlimited to: Restasis™, Hyalein™, Celluvisc™, Systane™, Tears Naturale™,Mytear®, Refresh Tears™, Refresh™, Genteal™, Viscotears™, Artelac™,Opelead™, Opti-Free Express™ Soft Lens Solutions, RENU™, Opti-Free™,Opti-Free Replenis™, AOSEPT™, RENU PLUS™, RENU MULTIPLUS™, SENSITIVEEYES™, BOSTON SIMPLUS™, LENSEPT™ OPTIFREE REPLENISH™, OpthalmicNonsteroidal Anti-Inflammatories such as ACULAR™ (KETOROLAC), VOLTAREN™(DICLOFENAC), NEVANAC™ (NEPAFENAC), XIBROM™, (BROMFENAC), ISTA PHARM™,(NIFLAN), PRANOPROFEN, INDOCOLLYRE, INDOMETACIN; preparations for thetreatment of non-specific conjunctivitis such as PATANOL™ (OLOPATADINE),LIVOSTIN™ (LEVOCABASTINE), ZADITEN™ (KETOTIFEN), PATADAY™ (OLOPATADINE),OPTIVAR™ (AZELASTINE), RELESTAT™ (EPINASTINE), OYXIN™ (TETRYZOLINE),INTAL (CROMOGLICIC ACID), RIZABEN (TRANILAST), CLEAR EYES™(NAPHAZOLINE), DACRYOSERUM™ (BORIC ACID), Allergic eye care products,such as Naphcon-A™, Visine-A™, Opcon A™, Clear Eyes ACR™, All Clear AR™,Vasocon-A™. Gas-enriched fluids produced in accordance with thedisclosed invention may also be used to decontaminate or wash awaycontaminants from a tissue, intact and/or ex vivo. Contaminants can bemore thoroughly cleaned away by the gas-enriched fluids, which mayprovide other therapeutic benefits.

Particular embodiments provided herein relate to a diffuser-processedgas-enriched fluid as defined herein, comprising: a fluid host material;an infusion material diffused into the host material; and optionally, atleast one cosmetic and/or therapeutic agent dispersed in the hostmaterial. In certain embodiments, the infusion material comprises oxygenmicro-bubbles in the host fluid, wherein the majority of themicro-bubbles are less than 0.2 microns, or preferably less than 0.1microns in size.

Gas-enriched fluids produced in accordance with the disclosed inventionmay also be used to decontaminate or wash away contaminants from atissue, intact and/or ex vivo. Contaminants can be more thoroughlycleaned away by the gas-enriched fluids, which may provide othertherapeutic benefits.

There are several art-recognized methods of gas-enriching liquids (suchas oxygen-enriching water). For example, a turbine aeration system canrelease air near a set of rotating blades of an impeller, which mixesthe air or oxygen with the water, or water can be sprayed into the airto increase its oxygen content. Additionally, other systems on themarket inject air or oxygen into the water and subject the water/gas toa large-scale vortex. Naturally occurring levels of oxygen in water aretypically no more than 10 ppm (parts per million), which is consideredto be a level of 100% dissolved oxygen. Tests on certain devices haveshown that under ideal conditions, the device can attain upwards ofapproximately 20 ppm, or twice the natural oxygen levels of water. Incertain embodiments, the oxygen level may be even higher.

Particular embodiments provided herein relate to a diffuser-processedgas-enriched fluid as defined herein, comprising: a fluid host material;an infusion material diffused into the host material; and optionally, atleast one cosmetic and/or therapeutic agent dispersed in the hostmaterial. In certain embodiments, the infusion material comprises oxygenmicro-bubbles in the host fluid, wherein the majority of themicro-bubbles are less than 0.2 microns, or preferably less than 0.1microns in size. In certain embodiments, the dissolved oxygen level inthe infused fluid host material may be maintained at greater than about30 ppm at atmospheric pressure for at least 13 hours. In otherparticular embodiments, the dissolved oxygen level in the infused fluidhost material may be maintained at greater than 40 ppm at atmosphericpressure for at least 3 hours.

In additional embodiments, the infused fluid host material furthercomprises a saline solution. In further embodiments, the infused fluidhost material maintains a dissolved oxygen level of at least about 20ppm to about 40 ppm for a period of at least 100 days, preferably atleast 365 days within a sealed container at atmospheric pressure. Incertain embodiments, the infused fluid host material may have adissolved oxygen level of at least 50 ppm at atmospheric pressure.

In certain embodiments, the infused fluid host material exhibitsRayleigh scattering for a laser beam shining therethrough for a selectedperiod of time after the oxygen has been diffused into therein.

Table 1 illustrates various partial pressure measurements taken in ahealing wound treated with an oxygen-enriched saline solution and insamples of the gas-enriched oxygen-enriched saline solution of thepresent invention.

TABLE 1 TISSUE OXYGEN MEASUREMENTS Probe Z082BO In air: 171 mmHg 23° C.Column Partial Pressure (mmHg) B1 32-36 B2 169-200 B3  20-180* B4 40-60*wound depth minimal, majority >150, occasional 20 s

In certain embodiments, the dissolved oxygen level in the infused fluidhost material may be maintained at greater than about 30 ppm atatmospheric pressure for at least about 13 hours. In other particularembodiments, the dissolved oxygen level in the infused fluid hostmaterial may be maintained at greater than about 40 ppm at atmosphericpressure for at least about 3 hours. In further embodiments, the infusedfluid host material maintains a dissolved oxygen level of at least about20 ppm for a period of at least about 100 days within a sealed containerat atmospheric pressure. In certain embodiments, the infused fluid hostmaterial may have a dissolved oxygen level of at least about 10 ppm, atleast about 15 ppm, at least about 20 ppm, at least about 25 ppm, atleast about 30 ppm, at least about 35 ppm, at least about 40 ppm, atleast about 45 ppm, at least about 50 ppm, at least about 55 ppm, atleast about 60 ppm, at least about 65 ppm, at least about 70 ppm, atleast about 75 ppm, at least about 77 ppm, at least about 80 ppm, atleast about 85 ppm, at least about 90 ppm, at least about 95 ppm, atleast about 100 ppm, or greater or any value therebetween, atatmospheric pressure.

In certain embodiments, the infused fluid host material exhibitsRayleigh scattering for a laser beam shining therethrough for a selectedperiod of time after the oxygen has been diffused therein. In certainimplementations, the infused fluid host material includes solvatedelectrons created in the solution by the process for diffusing theoxygen therein. In yet further embodiments, the infused fluid hostmaterial containing oxygen diffused therein is produced in anon-restricted flow through manner.

By using the diffuser device described herein with respect to exemplaryembodiments illustrated in the corresponding figures, an output fluidmay be achieved having a gas diffused therein that has a number ofcharacteristics and provides a number of advantages for use as atherapeutic composition. Solutions have been created using freshwater,saline, oxygen, nitrogen and other components. Experiments haveindicated that oxygen bubbles produced within saline solution aregenerally no greater than approximately 0.1 micron in size.

Bubble Size Measurements

Experimentation was performed to determine a size of the bubbles of gasdiffused within the fluid by the mixing device 100. While experimentswere not performed to measure directly the size of the bubbles,experiments were performed that established that the bubble size of themajority of the gas bubbles within the fluid was smaller than 0.1microns. In other words, the experiments determined a size thresholdvalue below which the sizes of the majority of bubbles fall.

This size threshold value or size limit was established by passing theoutput material 102 formed by processing a fluid and a gas in the mixingdevice 100 through a 0.22 filter and a 0.1 micron filter. In performingthese tests, a volume of the first material 110, in this case, a fluid,and a volume of the second material 120, in this case, a gas, werepassed through the mixing device 100 to generate a volume of the outputmaterial 102 (i.e., a fluid having a gas diffused therein). Sixtymilliliters of the output material 102 was drained into a 60 ml syringe.The DO level of the fluid within the syringe was then measured using anOrion 862a. The Orion 862a is capable of measuring DO levels within afluid. The fluid within the syringe was injected through a 0.22 micronfilter into a 50 ml beaker. The filter comprised the Milipor Millex GP50filter. The DO level of the material in the 50 ml beaker was thenmeasured. The experiment was performed three times to achieve theresults illustrated in Table 2 below.

TABLE 2 DO levels DO AFTER 0.22 MICRON DO IN SYRINGE FILTER 42.1 ppm39.7 ppm 43.4 ppm 42.0 ppm 43.5 ppm 39.5 ppm

As can be seen, the DO levels measured within the syringe and the DOlevels measured within the 50 ml beaker were not changed drastically bypassing the output material 102 through the 0.22 micron filter. Theimplication of this experiment is that the bubbles of dissolved gaswithin the output material 102 are not larger than 0.22 micronsotherwise there would be a significantly greater reduction in the DOlevels in the output material 102 passed through the 0.22 micron filter.

A second test was performed in which the 0.1 micron filter wassubstituted for the 0.22 micron filter. In this experiment, salinesolution was processed with oxygen in the mixing device 100 and a sampleof the output material 102 was collected in an unfiltered state. The DOlevel of the unfiltered sample was 44.7 ppm. The output material 102 wasfiltered using the 0.1 micron filter and two additional samples werecollected. The DO level of the first sample was 43.4 ppm. The DO levelof the second sample was 41.4 ppm. Then, the filter was removed and afinal sample was taken from the unfiltered output material 102. Thefinal sample had a DO level of 45.4 ppm. These results were consistentwith those seen using the Millipore 0.2 micron filter. These resultslead to the conclusion that there is a trivial reduction in the DOlevels of the output material 102 passed through the 0.1 micron filterproviding an indication that the majority of the bubbles in theprocessed saline solution are no greater than 0.1 micron in size.

As appreciated in the art, the double-layer (interfacial) (DL) appearson the surface of an object when it is placed into a liquid. Thisobject, for example, might be that of a solid surface (e.g., rotor andstator surfaces), solid particles, gas bubbles, liquid droplets, orporous body. In the mixing device 100, bubble surfaces represent asignificant portion of the total surface area present within the mixingchamber that may be available for electrokinetic double-layer effects.Therefore, in addition to the surface area and retention time aspectsdiscussed elsewhere herein, the relatively small bubble sizes generatedwithin the mixer 100 compared to prior art devices 10, may alsocontribute, at least to some extent, to the overall electrokineticeffects and output fluid properties disclosed herein. Specifically, inpreferred embodiments, as illustrated by the mixer 100, all of the gasis being introduced via apertures on the rotor (no gas is beingintroduced through stator apertures. Because the rotor is rotating at ahigh rate (e.g., 3,400 rpm) generating substantial shear forces at andnear the rotor surface, the bubble size of bubbles introduced via, andadjacent to the spinning rotor surface apertures would be expected to besubstantially (e.g., 2 to 3-times smaller) smaller than those introducedvia and near the stationary stator. The average bubble size of the priorart device 10 may, therefore, be substantially larger because at leasthalf of the gas is introduced into the mixing chamber from thestationary stator apertures. Because the surface area of a spheresurface varies with r², any such bubble component of the electrokineticsurface area of the mixing device 100 may be substantially greater thanthat of the prior art diffusion device 10.

Therefore, without being bound by theory, not only does the mixingchamber of the mixing device 100 have (i) a substantially higher surfaceto volume ratio than that of the prior art device 10 (the prior artdevice 10 has a ratio of surface to volume of 10.9, whereas the presentmixer 100 has a surface to volume ratio of 39.4), along with (ii) a7-fold greater dwell-time, but (iii) the unique properties of thecurrent output solutions may additionally reflect a contribution fromthe substantially larger bubble surface area in the mixing device 100.These distinguishing aspects reflect distinguishing features of thepresent mixer 100, and likely each contribute to the uniqueelectrokinetic properties of the inventive output materials/fluids.

Referring now to FIG. 30, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlthin-walled plastic bottle and a 1000 ml glass bottle out to at least365 days. Each of the bottles was capped and stored at 65° Fahrenheit.As can be seen in the Figure, the DO levels of the oxygen-enriched fluidremained fairly constant out to at least 365 days.

Referring to FIG. 31, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlplastic thin-walled bottle and a 1000 ml glass bottle. Both bottles wererefrigerated at 39° Fahrenheit. Again, DO levels of the oxygen-enrichedfluid remained steady and decreased only slightly out to at least 365days.

Compositions Comprising Hydrated (Solvated) Electrons Imparted to theInventive Compositions by the Inventive Processes

In certain embodiments as described herein (see under “Double-layer”),the gas-enriched fluid is generated by the disclosed electromechanicalprocesses in which molecular oxygen is diffused or mixed into the fluidand may operate to stabilize charges (e.g., hydrated (solvated)electrons) imparted to the fluid. Without being bound by theory ormechanism, certain embodiments of the present invention relate to aoxygen-enriched fluid (output material) comprising charges (e.g.,hydrated (solvated) electrons) that are added to the materials as thefirst material is mixed with oxygen in the inventive mixer device toprovide the combined output material. According to particular aspects,these hydrated (solvated) electrons (alternately referred to herein as‘solvated electrons’) are stabilized in the inventive solutions asevidenced by the persistence of assayable effects mediated by thesehydrated (solvated) electrons. Certain embodiments may relate tohydrated (solvated) electrons and/or water-electron structures,clusters, etc., (See, for example, Lee and Lee, Bull. Kor. Chem. Soc.2003, v. 24, 6; 802-804; 2003).

Horseradish Peroxidase (HRP) Effects.

Horseradish peroxidase (HRP) is isolated from horseradish roots(Amoracia rusticana) and belongs to the ferroprotoporphyrin group (Hemegroup) of peroxidases. HRP readily combines with hydrogen peroxide orother hydrogen donors to oxidize the pyrogallol substrate. Additionally,as recognized in the art, HRP facilitates autoxidative degradation ofindole-3-acetic acid in the absence of hydrogen peroxide (see, e.g.,Heme Peroxidases, H. Brian Dunford, Wiley-VCH, 1999, Chapter 6, pages112-123, describing that auto-oxidation involves a highly efficientbranched-chain mechanism; incorporated herein by reference in itsentirety). The HRP reaction can be measured in enzymatic activity units,in which Specific activity is expressed in terms of pyrogallol units.One pyrogallol unit will form 1.0 mg purpurogallin from pyrogallol in 20sec at pH 6.0 at 20° C. This purpurogallin (20 sec) unit is equivalentto approx. 18 μM units per min at 25° C.

It is known that Horseradish peroxidase enzyme catalyzes theauto-oxidation of pyrogallol by way of facilitating reaction with themolecular oxygen in a fluid. (Khajehpour et al., PROTEINS: Struct,Funct, Genet. 53: 656-666 (2003)). It is also known that oxygen bindsthe heme pocket of horseradish peroxidase enzyme through a hydrophobicpore region of the enzyme (between Phe68 and Phe142), whose conformationlikely determines the accessibility of oxygen to the interior. Accordingto particular aspects, and without being bound by mechanism, becausesurface charges on proteins are known in the protein art to influenceprotein structure, the solvated electrons present in the inventivegas-enriched fluid may act to alter the conformation of the horseradishperoxidase such that greater oxygen accessibility may result. Thegreater accessibility of oxygen to the prosthetic heme pocket of thehorseradish peroxidase enzyme may in turn allow for increased HRPreactivity, when compared with prior art oxygenated fluids(pressure-pot, fine-bubbled).

In any event, according to particular aspects, production of outputmaterial using the inventive methods and devices comprises a processinvolving: an interfacial double layer that provides a charge gradient;movement of the materials relative to surfaces pulling charge (e.g.,electrons) away from the surface by virtue of a triboelectric effect,wherein the flow of material produces a flow of solvated electrons.Moreover, according to additional aspects, and without being bound bymechanism, the orbital structure of diatomic oxygen creates chargeimbalances (e.g., the two unpaired electrons affecting the hydrogenbonding of the water) in the hydrogen bonding arrangement within thefluid material (water), wherein electrons are solvated and stabilizedwithin the imbalances.

Several chemical tests of the inventive oxygen-enriched fluid for thepresence of hydrogen peroxide were conducted as described below, andnone of these tests were positive (sensitivity of 0.1 ppm hydrogenperoxide). Thus, the inventive oxygen-enriched fluid of the instantapplication contain no, or less than 0.1 ppm hydrogen peroxide.

According to particular aspects, despite the absence of hydrogenperoxide, the inventive combination of oxygen-enrichment and solvatedelectrons imparted by the double-layer effects and configuration of thepresently claimed devices may act to alter the conformation and/or hemegroup accessibility of the horseradish peroxidase.

Glutathione Peroxidase Study

The inventive oxygen-enriched output fluid material was tested for thepresence of hydrogen peroxide by testing the reactivity with glutathioneperoxidase using a standard assay (Sigma). Water samples were tested byadding the enzymatic reagents. Continuous spectrophotometric ratedetermination was made at A₃₄₀ nm, and room temperature (25° Celsius).Samples tested were: 1. deionized water (negative control), 2. inventiveoxygen-enriched fluid at low concentration, 3. inventive oxygen-enrichedfluid at high concentration, 4. hydrogen peroxide (positive control).The hydrogen peroxide positive control showed a strong reactivity, whilenone of the other fluids tested reacted with the glutathione.

Device for Generating Gas-Enriched Fluids or Solutions

Description of the Related Art

FIG. 1 provides a partial block diagram, partial cross-sectional view ofa prior art device 10 for diffusing or emulsifying one or two gaseous orliquid materials (“infusion materials”) into another gaseous or liquidmaterial (“host material”) reproduced from U.S. Pat. No. 6,386,751,incorporated herein by reference in its entirety. The device 10 includesa housing configured to house a stator 30 and a rotor 12. The stator 30encompasses the rotor 12. A tubular channel 32 is defined between therotor 12 and the stator 30. The generally cylindrically shaped rotor 12has a diameter of about 7.500 inches and a length of about 6.000 inchesproviding a length to diameter ratio of about 0.8.

The rotor 12 includes a hollow cylinder, generally closed at both ends.A gap exists between each of the first and second ends of the rotor 12and a portion of the housing 34. A rotating shaft 14 driven by a motor18 is coupled to the second end of the rotor 12. The first end of therotor 12 is coupled to an inlet 16. A first infusion material passesthrough the inlet 16 and into the interior of the rotor 12. The firstinfusion material passes from the interior of the rotor 12 and into thechannel 32 through a plurality of openings 22 formed in the rotor 12.

The stator 30 also has openings 22 formed about its circumference. Aninlet 36 passes a second infusion material to an area 35 between thestator 30 and the housing 34. The second infusion material passes out ofthe area 35 and into the channel 32 through openings 22.

An external pump (not shown) is used to pump the host material into asingle inlet port 37. The host material passes through a single inletport 37 and into the channel 32 where it encounters the first and secondinfusion materials, which enter the channel 32 through openings 22. Theinfusion materials may be pressurized at their source to prevent thehost material from passing through openings 22.

The inlet port 37, is configured and positioned such that it is locatedalong only a relatively small portion (< about 5%) of the annular inletchannel 32, and is substantially parallel to the axis of rotation of therotor 12 to impart an axial flow toward a portion of the channel 32 intothe host material.

Unfortunately, before entering the tubular channel 32, the host materialmust travel in tortuous directions other than that of the axial flow(e.g., including in directions substantially orthogonal thereto) anddown into and between the gap formed between the first end of the rotor12 and the housing 34 (i.e., down a portion of the first end of therotor adjacent to the inlet 16 between the end of the rotor 12 and thehousing 34). The non-axial and orthogonal flow, and the presence of thehost material in the gap between the first end of the rotor 12 and thehousing 34 causes undesirable and unnecessary friction. Further, it ispossible for a portion of the host material to become trapped in eddycurrents swirling between the first end of the rotor and the housing.Additionally, in the device 10, the host material must negotiate atleast two right angles to enter any aspect of the annual of the annularinlet of the tubular channel 32.

A single outlet port 40 is formed in the housing 34. The combined hostmaterial and infusion material(s) exit the channel 32 via the outlet 40.The outlet port 40, which is also located along only a limited portion(< about 5%) of the annular outlet of tubular channel 32, issubstantially parallel to the axis of rotation of the rotor 12 to impartor allow for an axial flow of the combined materials away from thelimited portion of the annular outlet of tubular channel 32 into theoutlet port 40. An external pump 42 is used to pump the exiting fluidthrough the outlet port 40.

Unfortunately, before exiting the channel 32, a substantial portion ofthe exiting material must travel in a tortuous direction other than thatof the axial flow (e.g., including in directions substantiallyorthogonal thereto) and down into and between the gap formed between thesecond end of the rotor 12 and the housing 34 (i.e., down a portion ofthe second end of the rotor adjacent to the shaft 14 between the end ofthe rotor 12 and the housing 34). As mentioned above, the non-axial andorthogonal flow, and the presence of the host material in the other gapbetween the end (in this case, the second end) of the rotor 12 and thehousing 34 causes additional undesirable and unnecessary friction.Further, it is possible for a portion of the host material to becometrapped in eddy currents swirling between the second end of the rotorand the housing. Additionally, in the device 10, a substantial portionof the exiting combined material must negotiate at least two rightangles as it exits from the annular exit of the tubular channel 32 intothe outlet port 40.

As is apparent to those of ordinary skill in the art, the inlet port 37imparts only an axial flow to the host material. Only the rotor 21imparts a circumferential flow into the host material. Further, theoutlet port 40 imparts or provides for only an axial flow into theexiting material. Additionally, the circumferential flow velocity vectoris imparted to the material only after it enters the annular inlet 37 ofthe tubular channel 32, and subsequently the circumferential flow vectormust be degraded or eliminated as the material enters the exit port 40.There is, therefore, a need for a progressive circumferentialacceleration of the material as it passes in the axial direction throughthe channel 32, and a circumferential deceleration upon exit of thematerial from the channel 32. These aspects, in combination with thetortuous path that the material takes from the inlet port 37 to theoutlet port 40, create a substantial friction and flow resistance overthe path that is accompanied by a substantial pressure differential (26psi, at 60 gallons/min flow rate) between the inlet 37 and outlet 40ports, and these factors, inter alia, combine to reduce the overallefficiency of the system.

Electrokinetically Oxygen-Enriched Aqueous Fluids and Solutions

FIG. 2 provides a block diagram illustrating some of the components of amixing device 100 and the flow of material into, within, and out of thedevice. The mixing device 100 combines two or more input materials toform an output material 102, which may be received therefrom into astorage vessel 104. The mixing device 100 agitates the two or more inputmaterials in a novel manner to produce an output material 102 havingnovel characteristics. The output material 102 may include not only asuspension of at least one of the input materials in at least one of theother input materials (e.g., emulsions) but also a novel combination(e.g., electrostatic combinations) of the input materials, a chemicalcompound resulting from chemical reactions between the input materials,combinations having novel electrostatic characteristics, andcombinations thereof.

The input materials may include a first material 110 provided by asource 112 of the first material, a second material 120 provided by asource 122 of the second material, and optionally a third material 130provided by a source 132 of the third material. The first material 110may include a liquid, such as water, saline solution, chemicalsuspensions, polar liquids, non-polar liquids, colloidal suspensions,cell growing media, and the like. In some embodiments, the firstmaterial 110 may include the output material 102 cycled back into themixing device 100. The second material 120 may consist of or include agas, such as oxygen, nitrogen, carbon dioxide, carbon monoxide, ozone,sulfur gas, nitrous oxide, nitric oxide, argon, helium, bromine, andcombinations thereof, and the like. In preferred embodiments, the gas isor comprises oxygen. The optional third material 130 may include eithera liquid or a gas. In some embodiments, the third material 130 may be orinclude the output material 102 cycled back into the mixing device 100(e.g., to one or more of the pumps 210, 220 or 230, and/or into thechamber 310, and/or 330).

Optionally, the first material 110, the second material 120, and theoptional third material 130 may be pumped into the mixing device 100 byan external pump 210, an external pump 220, and an external pump 230,respectively. Alternatively, one or more of the first material 110, thesecond material 120, and the optional third material 130 may be storedunder pressure in the source 112, the source 122, and the source 132,respectively, and may be forced into the mixing device 100 by thepressure. The invention is not limited by the method used to transferthe first material 110, the second material 120, and optionally, thethird material 130 into the mixing device 100 from the source 112, thesource 122, and the source 132, respectively.

The mixing device 100 includes a first chamber 310 and a second chamber320 flanking a mixing chamber 330. The three chambers 310, 320, and 330are interconnected and form a continuous volume.

The first material 110 is transferred into the first chamber 310 andflows therefrom into the mixing chamber 330. The first material 110 inthe first chamber 310 may be pumped into the first chamber 310 by aninternal pump 410. The second material 120 is transferred into themixing chamber 330. Optionally, the third material 130 may betransferred into the mixing chamber 330. The materials in the mixingchamber 330 are mixed therein to form the output material 102. Then, theoutput material 102 flows into the second chamber 320 from which theoutput material 102 exits the mixing device 100. The output material 102in the mixing chamber 330 may be pumped into the second chamber 320 byan internal pump 420. Optionally, the output material 102 in the secondchamber 320 may be pumped therefrom into the storage vessel 104 by anexternal pump 430 (e.g., alone or in combination with the internal pump410 and/or 420).

In particular aspects, a common drive shaft 500 powers both the internalpump 410 and the internal pump 420. The drive shaft 500 passes throughthe mixing chamber 330 and provides rotational force therein that isused to mix the first material 110, the second material 120, andoptionally, the third material 130 together. The drive shaft 500 ispowered by a motor 510 coupled thereto.

FIG. 3 provides a system 512 for supplying the first material 110 to themixing device 100 and removing the output material 102 from the mixingdevice 100. In the system 512, the storage vessel 104 of the outputmaterial 102 and the source 112 of the first material 110 are combined.The external pump 210 is coupled to the combined storage vessel 104 andsource 112 by a fluid conduit 514 such as hose, pipe, and the like. Theexternal pump 210 pumps the combined first material 110 and outputmaterial 102 from the combined storage vessel 104 and source 112 throughthe fluid conduit 514 and into a fluid conduit 516 connecting theexternal pump 210 to the mixing device 100. The output material 102exits the mixing device 100 through a fluid conduit 518. The fluidconduit 518 is coupled to the combined storage vessel 104 and source 112and transports the output material 102 exiting the mixing device 100 tothe combined storage vessel 104 and source 112. The fluid conduit 518includes a valve 519 that establishes an operating pressure or backpressure within the mixing device 100.

Referring to FIGS. 2 and 4-9 and 11, a more detailed description ofvarious components of an embodiment of the mixing device 100 will beprovided. The mixing device 100 is scalable. Therefore, dimensionsprovided with respect to various components may be used to construct anembodiment of the device or may be scaled to construct a mixing deviceof a selected size.

Turning to FIG. 4, the mixing device 100 includes a housing 520 thathouses each of the first chamber 310, the mixing chamber 330, and thesecond chamber 320. As mentioned above, the mixing device 100 includesthe drive shaft 500, which rotates during operation of the device.Therefore, the mixing device 100 may vibrate or otherwise move.Optionally, the mixing device 100 may be coupled to a base 106, whichmay be affixed to a surface such as the floor to maintain the mixingdevice 100 in a substantially stationary position.

The housing 520 may be assembled from two or more housing sections. Byway of example, the housing 520 may include a central section 522flanked by a first mechanical seal housing 524 and a second mechanicalseal housing 526. A bearing housing 530 may be coupled to the firstmechanical seal housing 524 opposite the central section 522. A bearinghousing 532 may be coupled to the second mechanical seal housing 526opposite the central section 522. Optionally, a housing section 550 maybe coupled to the bearing housings 530.

Each of the bearing housings 530 and 532 may house a bearing assembly540 (see FIGS. 5 and 6). The bearing assembly 540 may include anysuitable bearing assembly known in the art including a model number“202SZZST” manufactured by SKF USA Inc, of Kulpsville, Pa., operating awebsite at www.skf.com.

Seals may be provided between adjacent housing sections. For example,o-ring 560 (see FIG. 5) may be disposed between the housing section 550and the bearing housing 530, o-ring 562 (see FIG. 5) may be disposedbetween the first mechanical seal housing 524 and the central section522, and o-ring 564 (see FIG. 6) may be disposed between the secondmechanical seal housing 526 and the central section 522.

Mixing Chamber 330

Turning now to FIG. 7, the mixing chamber 330 is disposed inside thecentral section 522 of the housing 520 between the first mechanical sealhousing 524 and the second mechanical seal housing 526. The mixingchamber 330 is formed between two components of the mixing device 100, arotor 600 and a stator 700. The rotor 600 may have a sidewall 604 withan inside surface 605 defining a generally hollow inside portion 610 andan outside surface 606. The sidewall 604 may be about 0.20 inches toabout 0.75 inches thick. In some embodiments, the sidewall 604 is about0.25 inches thick. However, because the mixing device 100 may be scaledto suit a particular application, embodiments of the device having asidewall 604 that is thicker or thinner than the values provided arewithin the scope of the present teachings. The sidewall 604 includes afirst end portion 612 and a second end portion 614 and a plurality ofthrough-holes 608 formed between the first end portion 612 and thesecond end portion 614. Optionally, the outside surface 606 of thesidewall 604 may include other features such as apertures, projections,textures, and the like. The first end portion 612 has a relieved portion616 configured to receive a collar 618 and the second end portion 614has a relieved portion 620 configured to receive a collar 622.

The rotor 600 is disposed inside the stator 700. The stator 700 has asidewall 704 with an inside surface 705 defining a generally hollowinside portion 710 into which the rotor 600 is disposed. The sidewall704 may be about 0.1 inches to about 0.3 inches thick. In someembodiments, the sidewall 604 is about 1.5 inches thick. The stator 700may be non-rotatably coupled to the housing 520 in a substantiallystationary position. Alternatively, the stator 700 may integrally formedwith the housing 520. The sidewall 704 has a first end portion 712 and asecond end portion 714. Optionally, a plurality of apertures 708 areformed in the sidewall 704 of the stator 700 between the first endportion 712 and the second end portion 714. Optionally, the insidesurface 705 of the sidewall 704 may include other features such asthrough-holes, projections, textures, and the like.

The rotor 600 rotates with respect to the stationary stator 700 about anaxis of rotation “α” in a direction indicated by arrow “C3” in FIG. 9.Each of the rotor 600 and the stator 700 may be generally cylindrical inshape and have a longitudinal axis. The rotor 600 has an outer diameter“D1” and the stator 700 may have an inner diameter “D2.” The diameter“D1” may range, for example, from about 0.5 inches to about 24 inches.In some embodiments, the diameter“D1” is about 3.04 inches. In someembodiments, the diameter “D1” is about 1.7 inches. The diameter “D2,”which is larger than the diameter “D1,” may range from about 0.56 inchesto about 24.25 inches. In some embodiments, the diameter “D2” is about 4inches. Therefore, the mixing chamber 330 may have a ring-shapedcross-sectional shape that is about 0.02 inches to about 0.125 inchesthick (i.e., the difference between the diameter “D2” and thediameter“D1”). In particular embodiments, the mixing chamber 330 isabout 0.025 inches thick. The channel 32 between the rotor 12 and thestator 34 of prior art device 10 (see FIG. 1) has a ring-shapedcross-sectional shape that is about 0.09 inches thick. Therefore, inparticular embodiments, the thickness of the mixing chamber 330 is lessthan about one third of the channel 32 of the prior art device 10.

The longitudinal axis of the rotor 600 may be aligned with its axis ofrotation “α.” The longitudinal axis of the rotor 600 may be aligned withthe longitudinal axis of the stator 700. The rotor 600 may have a lengthof about 3 inches to about 6 inches along the axis of rotation “α.” Insome embodiments, the rotor 600 may have a length of about 5 inchesalong the axis of rotation “α.” The stator 700 may have a length ofabout 3 inches to about 6 inches along the axis of rotation “α.” In someembodiments, the stator 700 may have a length of about 5 inches alongthe axis of rotation “α.”

While the rotor 600 and the stator 700 have been depicted as having agenerally cylindrical shape, those of ordinary skill in the artappreciate that alternate shapes may be used. For example, the rotor 600and the stator 700 may be conically, spherically, arbitrarily shaped,and the like. Further, the rotor 600 and the stator 700 need not beidentically shaped. For example, the rotor 600 may be cylindricallyshaped and the stator 700 rectangular shaped or vise versa.

The apertures 708 of the stator 700 and the through-holes 608 depictedin FIGS. 4-7 are generally cylindrically shaped. The diameter of thethrough-holes 608 may range from about 0.1 inches to about 0.625 inches.The diameter of the apertures 708 may range from about 0.1 inches toabout 0.625 inches. One or more of apertures 708 of the stator 700 mayhave a diameter that differs from the diameters of the other apertures708. For example, the apertures 708 may increase in diameter from thefirst end portion 712 of the stator 700 to the second end portion 714 ofthe stator 700, the apertures 708 may decrease in diameter from thefirst end portion 712 of the stator 700 to the second end portion 714 ofthe stator 700, or the diameters of the apertures 708 may vary inanother manner along the stator 700. One or more of through-holes 608 ofthe rotor 600 may have a diameter that differs from the diameters of theother through-holes 608. For example, the through-holes 608 may increasein diameter from the first end portion 612 of the rotor 600 to thesecond end portion 614 of the rotor 600, the through-holes 608 maydecrease in diameter from the first end portion 612 of the rotor 600 tothe second end portion 614 of the rotor 600, or the diameters of thethrough-holes 608 may vary in another manner along the rotor 600.

As described below with reference to alternate embodiments, theapertures 708 and the through-holes 608 may have shapes other thangenerally cylindrical and such embodiments are within the scope of thepresent invention. For example, the through-holes 608 may include anarrower portion, an arcuate portion, a tapered portion, and the like.Referring to FIG. 7, each of the through-holes 608 includes an outerportion 608A, a narrow portion 608B, and a tapered portion 608Cproviding a transition between the outer portion 608A and the narrowportion 608B. Similarly, the apertures 708 may include a narrowerportion, an arcuate portion, a tapered portion, and the like.

FIG. 8 provides a non-limiting example of a suitable arrangement of theapertures 708 of the stator 700 and the through-holes 608 of the rotor600. The apertures 708 of the stator 700 may be arranged insubstantially parallel lateral rows “SLAT-1” through “SLAT-6”substantially orthogonal to the axis of rotation “α.” The apertures 708of the stator 700 may also be arranged in substantially parallellongitudinal rows “SLONG-1” through “SLONG-7” substantially parallelwith the axis of rotation “α.” In other words, the apertures 708 of thestator 700 may be arranged in a grid-like pattern of orthogonal rows(i.e., the lateral rows are orthogonal to the longitudinal rows) havingthe longitudinal rows “SLONG-1” through “SLONG-7” substantially parallelwith the axis of rotation “α.”

Like the apertures 708 of the stator 700, the through-holes 608 of therotor 600 may be arranged in substantially parallel lateral rows“RLAT-1” through “RLAT-6” substantially orthogonal to the axis ofrotation “α.” However, instead of being arranged in a grid-like patternof orthogonal rows, the through-holes 608 of the rotor 600 may also bearranged in substantially parallel rows “RLONG-1” through “RLONG-7” thatextend longitudinally along a helically path. Alternatively, thethrough-holes 608 of the rotor 600 may also be arranged in substantiallyparallel rows “RLONG-1” through “RLONG-7” that extend longitudinally atan angle other than parallel with the axis of rotation “α.”

The apertures 708 of the stator 700 and the through-holes 608 of therotor 600 may be configured so that when the rotor 600 is disposedinside the stator 700 the lateral rows “SLAT-1” to “SLAT-6” at leastpartially align with the lateral rows “RLAT-1” to “RLAT-6,”respectively. In this manner, as the rotor 600 rotates inside the stator700, the through-holes 608 pass by the apertures 708.

The through-holes 608 in each of the lateral rows “RLAT-1” to “RLAT-6”may be spaced apart laterally such that all of the through-holes 608 inthe lateral row align, at least partially, with the apertures 708 in acorresponding one of the lateral rows “SLAT-1” to “SLAT-6” of the stator700 at the same time. The longitudinally extending rows “RLONG-1”through “RLONG-6” may be configured such that the through-holes 608 inthe first lateral row “RLAT-1” in each of the longitudinally extendingrows passes completely by the apertures 708 of the corresponding lateralrow “SLAT-1” before the through-holes 608 in the last lateral row“RLAT-6” begin to partially align with the apertures 708 of thecorresponding last lateral row “SLAT-6” of the stator 700.

While, in FIG. 8, six lateral rows and six longitudinally extending rowshave been illustrated with respect to the rotor 600 and six lateral rowsand seven longitudinally extending rows have been illustrated withrespect stator 700, it is apparent to those of ordinary skill in the artthat alternate numbers of lateral rows and/or longitudinal rows may beused with respect to the rotor 600 and/or stator 700 without departingfrom the present teachings.

To ensure that only one pair of openings between corresponding lateralrows will be coincident at any one time, the number of apertures 708 ineach of the lateral rows “SLAT-1” to “SLAT-6” on the stator 700 maydiffer by a predetermined number (e.g., one, two, and the like) thenumber of through-holes 608 in each of the corresponding lateral rows“RLAT-1” to “RLAT-6” on the rotor 600. Thus, for example, if lateralrow“RLAT-1” has twenty through-holes 608 evenly spaced around thecircumference of rotor 600, the lateral row “SLAT-1” may have twentyapertures 708 evenly spaced around the circumference of stator 700.

Returning to FIG. 7, the mixing chamber 330 has an open first endportion 332 and an open second end portion 334. The through-holes 608formed in the sidewall 604 of the rotor 600 connect the inside portion610 of the rotor 600 with the mixing chamber 330.

The rotor 600 is rotated inside the stator 700 by the drive shaft 500aligned with the axis of rotation “α” of the rotor 600. The drive shaft500 may be coupled to the first end portion 612 and the second endportion 614 of the rotor 600 and extend through its hollow insideportion 610. In other words, a portion 720 of the drive shaft 500 isdisposed in the hollow inside portion 610 of the rotor 600.

The collar 618 is configured to receive a portion 721 of the drive shaft500 disposed in the hollow inside portion 610 and the collar 622 isconfigured to receive a portion 722 of the drive shaft 500 disposed inthe hollow inside portion 610.

The portion 721 has an outer diameter “D3” that may range from about 0.5inches to about 2.5 inches. In some embodiments, the diameter “D3” isabout 0.625 inches. The portion 722 has an outer diameter “D4” that maybe substantially similar to the diameter “D3,” although, this is notrequired. The diameter “D4” may range from about 0.375 inches to about2.5 inches.

The rotor 600 may be non-rotationally affixed to the portion 721 and theportion 722 of the drive shaft 500 by the collar 618 and the collar 622,respectively. By way of example, each of the collars 618 and 622 may beinstalled inside relieved portions 616 and 620, respectively. Then, thecombined rotor 600 and collars 618 and 622 may be heated to expand them.Next, the drive shaft 500 is inserted through the collars 618 and 622and the assembly is allowed to cool. As the collars 618 and 622 shrinkduring cooling, they tighten around the portions 722A and 722B of thedrive shaft 500, respectively, gripping it sufficiently tightly toprevent the drive shaft 500 from rotating relative to the rotor 600. Thecollar 618, which does not rotate with respect to either the portion 721or the relieved portion 616, translates the rotation of the drive shaft500 to the first end portion 612 the rotor 600. The collar 622, whichdoes not rotate with respect to either the portion 722 or the relievedportion 620, translates the rotation of the drive shaft 500 to thesecond end portion 614 of the rotor 600. The drive shaft 500 and therotor 600 rotate together as a single unit.

The drive shaft 500 may have a first end portion 724 (see FIG. 5) and asecond end portion 726 (see FIG. 6). The first end portion 724 may havea diameter “D5” of about 0.5 inches to about 1.75 inches. In particularembodiments, the diameter “D5” may be about 1.25 inches. The second endportion 726 may have a diameter “D6” that may be substantially similarto diameter “D5.”

The second material 120 may be transported into the mixing chamber 330through one of the first end portion 724 and the second end portion 726of the rotating drive shaft 500. The other of the first end portion 724and the second end portion 726 of the drive shaft 500 may be coupled tothe motor 510. In the embodiment depicted in FIGS. 5 and 6, the secondmaterial 120 is transported into the mixing chamber 330 through thefirst end portion 724 and the second end portion 726 of the drive shaft500 is coupled to the motor 510.

Turning to FIG. 5, the drive shaft 500 may have a channel 728 formedtherein that extends from first end portion 724 into the portion 720disposed in the inside portion 610 of the rotor 600. The channel 728 hasan opening 730 formed in the first end portion 724. When the mixingdevice 100 is operating, the second material 120 is introduced into thechannel 728 through the opening 730.

A valve 732 may be disposed inside a portion of the channel 728 locatedin the first end portion 724 of the drive shaft 500. The valve 732 mayrestrict or otherwise control the backward flow of the second material120 from inside the hollow inside portion 610 through the channel 728and/or the forward flow of the second material 120 into the channel 728.The valve 732 may include any valve known in the art including a checkvalve. A suitable check valve includes a part number “CKFA1876205A,”free flow forward check valve, manufactured by The Lee Company USAhaving an office in Bothell, Wash. and operating a website atwww.theleeco.com.

The drive shaft 500 may include an aperture 740 located in the insideportion 610 of the rotor 600 that connects the channel 728 with theinside portion 610 of the rotor 600. While only a single aperture 740 isillustrated in FIG. 5, it is apparent to those of ordinary skill in theart that multiple apertures may be used to connect the channel 728 withthe inside portion 610 of the rotor 600.

Referring to FIG. 2, optionally, the external pump 220 may pump thesecond material 120 into the mixing device 100. The pump 220 may includeany suitable pump known in the art. By way of non-limiting example, thepump 220 may include any suitable pump known in the art including adiaphragm pump, a chemical pump, a peristaltic pump, a gravity fed pump,a piston pump, a gear pump, a combination of any of the aforementionedpumps, and the like. If the second material 120 is a gas, the gas may bepressurized and forced into the opening 730 formed in the first endportion 724 of the drive shaft 500 by releasing the gas from the source122.

The pump 220 or the source 122 is coupled to the channel 728 by thevalve 732. The second material 120 transported inside the channel 728exits the channel 728 into the inside portion 610 of the rotor 600through the aperture 740. The second material 120 subsequently exits theinside portion 610 of the rotor 600 through the through-holes 608 formedin the sidewall 608 of the rotor 600.

Referring to FIG. 5, the mixing device 100 may include a seal assembly750 coupled to the first end portion 724 of the drive shaft 500. Theseal assembly 750 is maintained within a chamber 752 defined in thehousing 520. The chamber 752 has a first end portion 754 spaced acrossthe chamber from a second end portion 756. The chamber 752 also includesan input port 758 and an output port 759 that provide access into thechamber 752. The chamber 752 may be defined by housing section 550 andthe bearing housing 530. The first end portion 754 may be formed in thehousing section 550 and the second end portion 756 may be adjacent tothe bearing housing 530. The input port 758 may be formed in the bearinghousing 530 and the output port 759 may be formed in the housing section550.

The seal assembly 750 includes a first stationary seal 760 installed inthe first end portion 754 of the chamber 752 in the housing section 550and the bearing housing 530. The first stationary seal 760 extendsaround a portion 762 of the first end portion 724 of the drive shaft500. The seal assembly 750 also includes a second stationary seal 766installed in the second end portion 756 of the chamber 752 in thebearing housing 530. The second stationary seal 766 extends around aportion 768 of the first end portion 724 of the drive shaft 500.

The seal assembly 750 includes a rotating assembly 770 that isnon-rotatably coupled to the first end portion 724 of the drive shaft500 between the portion 762 and the portion 768. The rotating assembly770 rotates therewith as a unit. The rotating assembly 770 includes afirst seal 772 opposite a second seal 774. A biasing member 776 (e.g., aspring) is located between the first seal 772 and the second seal 774.The biasing member 776 biases the first seal 772 against the firststationary seal 760 and biases the second seal 774 against the secondstationary seal 766.

A cooling lubricant is supplied to the chamber 752 and around rotatingassembly 770. The lubricant enters the chamber 752 through the inputport 758 and exits the chamber 752 through output port 759. Thelubricant may lubricate the bearing assembly 540 housed by the bearinghousing 530. A chamber 570 may be disposed between the bearing housing530 and the mechanical seal housing 524. The bearing housing 530 mayalso include a second input port 759 connected to the chamber 570 intowhich lubricant may be pumped. Lubricant pumped into the chamber 570 maylubricate the bearing assembly 540. The seal assembly 750 maysignificantly, if not greatly, reduce frictional forces within thisportion of the device caused by the rotation of the rotor 600 and mayincrease the active life of the seals 770. The seals may includesurfaces constructed using silicon carbide.

Referring to FIG. 9, as the rotor 600 rotates about the axis of rotation“α” in the direction indicated by arrow “C1,” the rotor expels thesecond material 120 into the mixing chamber 330. The expelled bubbles,droplets, particles, and the like of the second material 120 exit therotor 600 and are imparted with a circumferential velocity (in adirection indicated by arrow “C3”) by the rotor 600. The second material120 may forced from the mixing chamber 330 by the pump 220 (see FIG. 2),the centrifugal force of the rotating rotor 600, buoyancy of the secondmaterial 120 relative to the first material 110, and a combinationthereof.

Motor 510

Returning to FIG. 6, the second end portion 726 of the drive shaft 500may be coupled to a rotating spindle 780 of a motor 510 by a coupler900. The spindle 780 may have a generally circular cross-sectional shapewith a diameter “D7” of about 0.25 inches to about 2.5 inches. Inparticular embodiments, the diameter “D7” may be about 0.25 inches toabout 1.5 inches. While in the embodiment depicted in FIG. 6, thediameter “D5” of the first end portion 724 of the drive shaft 500 issubstantially equal to the diameter “D7” and the spindle 780,embodiments in which one of the diameter “D5” and the diameter “D7” islarger than the other are within the scope of the present invention.

Referring also to FIG. 4, it may be desirable to cover or shield thecoupler 900. In the embodiment illustrated in FIGS. 4 and 6, a driveguard 910 covers the coupler 900. The drive guard 910 may be generallyU-shaped having a curved portion 914 flanked by a pair of substantiallylinear portions 915 and 916. The distal end of each of the substantiallylinear portions 915 and 916 of the drive guard 910 may have a flange 918and 919, respectively. The drive guard 910 may be fastened by each ofits flanges 918 and 919 to the base 106.

The motor 510 may be supported on the base 106 by a support member 920.The support member 920 may be coupled to the motor 510 near the spindle780. In the embodiment depicted, the support member 920 includes athrough-hole through which the spindle 780 passes. The support member920 may be coupled to the motor 510 using any method known in the art,including bolting the support member 920 to the motor 510 with one ormore bolts 940.

The coupler 900 may include any coupler suitable for transmitting asufficient amount of torque from the spindle 780 to the drive shaft 500to rotate the rotor 600 inside to the stator 700. In the embodimentillustrated in FIGS. 4 and 6, the coupler 900 is a bellows coupler. Abellows coupler may be beneficial if the spindle 780 and the drive shaft500 are misaligned. Further, the bellows coupler may help absorb axialforces exerted on the drive shaft 500 that would otherwise be translatedto the spindle 780. A suitable bellows coupler includes a model“BC32-8-8-A,” manufactured by Ruland Manufacturing Company, Inc. ofMarlborough, Mass., which operates a website at www.ruland.com.

The motor 510 may rotate the rotor 600 at about 0.1 revolutions perminute (“rpm”) to about 7200 rpm. The motor 510 may include any motorsuitable for rotating the rotor 600 inside to the stator 700 inaccordance with the present teachings. By way of non-limiting example, asuitable motor may include a one-half horsepower electric motor,operating at 230/460 volts and 3450 per minute (“rpm”). A suitable motorincludes a model “C4T34NC4C” manufactured by LEESON Electric Corporationof Grafton, Wis., which operates a website at www.leeson.com.

First Chamber 310

Turning to FIGS. 4 and 7, the first chamber 320 is disposed inside thecentral section 522 of the housing 520 between the first mechanical sealhousing 524 and the first end portions 612 and 712 of the rotor 600 andthe stator 700, respectively. The first chamber 310 may be annular andhave a substantially circular cross-sectional shape. The first chamber310 and the mixing chamber 330 form a continuous volume. A portion 1020of the drive shaft 500 extends through the first chamber 310.

As may best be viewed in FIG. 4, the first chamber 310 has an input port1010 through which the first material 110 enters the mixing device 100.The first material 110 may be pumped inside the first chamber 310 by theexternal pump 210 (see FIG. 2). The external pump 210 may include anypump known in the art for pumping the first material 110 at a sufficientrate to supply the first chamber 310.

The input port 1010 is oriented substantially orthogonally to the axisof rotation “α.” Therefore, the first material 110 enters the firstchamber 310 with a velocity tangential to the portion 1020 of the driveshaft 500 extending through the first chamber 310. The tangentialdirection of the flow of the first material 110 entering the firstchamber 310 is identified by arrow “T1.” In the embodiment depicted inFIGS. 4 and 7, the input port 1010 may be offset from the axis ofrotation “α.” As is apparent to those of ordinary skill in the art, thedirection of the rotation of the drive shaft 500 (identified by arrow“C1” in FIG. 9), has a tangential component. The input port 1010 ispositioned so that the first material 110 enters the first chamber 310traveling in substantially the same direction as the tangentialcomponent of the direction of rotation of the drive shaft 500.

The first material 110 enters the first chamber 310 and is deflected bythe inside of the first chamber 310 about the portion 1020 of the driveshaft 500. In embodiments wherein the first chamber 310 has asubstantially circular cross-sectional shape, the inside of the firstchamber 310 may deflect the first material 110 in a substantiallycircular path (identified by arrow “C2” in FIG. 9) about the portion1020 of the drive shaft 500. In such an embodiment, the tangentialvelocity of the first material 110 may cause it to travel about the axisof rotation “α” at a circumferential velocity, determined at least inpart by the tangential velocity.

Once inside the first chamber 310, the first material 110 may be pumpedfrom the first chamber 310 into the mixing chamber 330 by the pump 410residing inside the first chamber 310. In embodiments that include theexternal pump 210 (see FIG. 2), the external pump 210 may be configuredto pump the first material 110 into the first chamber 310 at a rate atleast as high as a rate at which the pump 410 pumps the first material110 from the first chamber 310.

The first chamber 310 is in communication with the open first endportion 332 of the mixing chamber 330 and the first material 110 insidethe first chamber 310 may flow freely into the open first end portion332 of the mixing chamber 330. In this manner, the first material 110does not negotiate any corners or bends between the mixing chamber 330and the first chamber 310. In the embodiment depicted, the first chamber310 is in communication with the entire open first end portion 332 ofthe mixing chamber 330. The first chamber 310 may be filled completelywith the first material 110.

The pump 410 is powered by the portion 1020 of the drive shaft 500extending through the first chamber 310. The pump 410 may include anypump known in the art having a rotating pump member 2022 housed inside achamber (i.e., the first chamber 310) defined by a stationary housing(i.e., the housing 520). Non-limiting examples of suitable pumps includerotary positive displacement pumps such as progressive cavity pumps,single screw pumps (e.g., Archimedes screw pump), and the like.

The pump 410 depicted in FIGS. 7 and 9, is generally referred to as asingle screw pump. In this embodiment, the pump member 2022 includes acollar portion 2030 disposed around the portion 1020 of the drive shaft500. The collar portion 2030 rotates with the portion 1020 of the driveshaft 500 as a unit. The collar portion 2030 includes one or more fluiddisplacement members 2040. In the embodiment depicted in FIGS. 7 and 9,the collar portion 2030 includes a single fluid displacement member 2040having a helical shape that circumscribes the collar portion 2030 alonga helical path.

Referring to FIG. 9, the inside of the first chamber 310 is illustrated.The pump 410 imparts an axial flow (identified by arrow “A1” and arrow“A2”) in the first material 110 inside the first chamber 310 toward theopen first end portion 332 of the mixing chamber 330. The axial flow ofthe first material 110 imparted by the pump 410 has a pressure that mayexceed the pressure obtainable by the external pump of the prior artdevice 10 (see FIG. 1).

The pump 410 may also be configured to impart a circumferential flow(identified by arrow “C2”) in the first material 110 as it travelstoward the open first end portion 332 of the mixing chamber 330. Thecircumferential flow imparted in the first material 110 before it entersthe mixing chamber 330 causes the first material 110 to enter the mixingchamber 330 already traveling in the desired direction at an initialcircumferential velocity. In the prior art device 10 depicted in FIG. 1,the first material 110 entered the channel 32 of the prior art device 10without a circumferential velocity. Therefore, the rotor 12 of the priorart device 10 alone had to impart a circumferential flow into the firstmaterial 110. Because the first material 110 is moving axially, in theprior art device 10, the first material 110 traversed at least a portionof the channel 32 formed between the rotor 12 and the stator 30 at aslower circumferential velocity than the first material 110 traversesthe mixing chamber 330 of the mixing device 100. In other words, if theaxial velocity of the first material 110 is the same in both the priorart device 10 and the mixing device 100, the first material 110 maycomplete more revolutions around the rotational axis “α” beforetraversing the axial length of the mixing chamber 330, than it wouldcomplete before traversing the axial length of the channel 32. Theadditional revolutions expose the first material 110 (and combined firstmaterial 110 and second material 120) to a substantially larger portionof the effective inside surface 706 (see FIG. 7) of the stator 700.

In embodiments including the external pump 210 (see FIG. 2), thecircumferential velocity imparted by the external pump 210 combined withthe input port 1010 being oriented according to the present teachings,may alone sufficiently increase the revolutions of the first material110 (and combined first material 110 and second material 120) about therotational axis “α.” Further, in some embodiments, the circumferentialvelocity imparted by the pump 210 and the circumferential velocityimparted by the pump 410 combine to achieve a sufficient number ofrevolutions of the first material 110 (and combined first material 110and second material 120) about the rotational axis “α.” As isappreciated by those of ordinary skill in the art, other structuralelements such as the cross-sectional shape of the first chamber 310 maycontribute to the circumferential velocity imparted by the pump 210, thepump 410, and a combination thereof.

In an alternate embodiment depicted in FIG. 10, the pump 410 may includeone or more vanes 2042 configured to impart a circumferential flow inthe first material 110 as it travels toward the open first end portion332 of the mixing chamber 330.

Second Chamber 320

Turning now to FIGS. 4 and 7, the second chamber 320 is disposed insidethe central section 522 of the housing 520 between the second mechanicalseal housing 526 and the second end portions 614 and 714 of the rotor600 and the stator 700, respectively. The second chamber 320 may besubstantially similar to the first chamber 310. However, instead of theinput port 1010, the second chamber 320 may include an output port 3010.A portion 3020 of the drive shaft 500 extends through the second chamber320.

The second chamber 320 and the mixing chamber 330 form a continuousvolume. Further, the first chamber 310, the mixing chamber 330, and thesecond chamber 320 form a continuous volume. The first material 110flows through the mixing device 100 from the first chamber 310 to themixing chamber 330 and finally to the second chamber 320. While in themixing chamber 330, the first material 110 is mixed with the secondmaterial 120 to form the output material 102. The output material 102exits the mixing device 100 through the output port 3010. Optionally,the output material 102 may be returned to the input port 1010 and mixedwith an additional quantity of the second material 120, the thirdmaterial 130, or a combination thereof.

The output port 3010 is oriented substantially orthogonally to the axisof rotation “α” and may be located opposite the input port 1010 formedin the first chamber 310. The output material 102 enters the secondchamber 320 from the mixing chamber 330 having a circumferentialvelocity (in the direction indicated by arrow “C3” in FIG. 9) impartedthereto by the rotor 600. The circumferential velocity is tangential tothe portion 3020 of the drive shaft 500 extending through the secondchamber 320. In the embodiment depicted in FIGS. 4, 6, and 7, the outputport 3010 may be offset from the axis of rotation “α.” The output port3010 is positioned so that the output material 102, which enters thesecond chamber 320 traveling in substantially the same direction inwhich the drive shaft 500 is rotating (identified in FIG. 9 by arrow“C1”), is traveling toward the output port 3010.

The output material 102 enters the second chamber 320 and is deflectedby the inside of the second chamber 320 about the portion 3020 of thedrive shaft 500. In embodiments wherein the second chamber 320 has asubstantially circular cross-sectional shape, the inside of the secondchamber 320 may deflect the output material 102 in a substantiallycircular path about the portion 3020 of the drive shaft 500.

Referring to FIG. 2, optionally, the output material 102 may be pumpedfrom inside the second chamber 320 by the external pump 430. Theexternal pump 430 may include any pump known in the art for pumping theoutput material 102 at a sufficient rate to avoid limiting throughput ofthe mixing device 100. In such an embodiment, the external pump 430 mayintroduce a tangential velocity (in a direction indicated by arrow “T2”in FIGS. 4 and 11) to at least a portion of the output material 102 asthe external pump 430 pumps the output material 102 from the secondchamber 320. The tangential velocity of the portion of the outputmaterial 102 may cause it to travel about the axis of rotation “α” at acircumferential velocity, determined in part by the tangential velocity.

Pump 420

Turning to FIGS. 6 and 7, the pump 420 residing inside the secondchamber 320 may pump the output material 102 from the second chamber 320into the output port 3010 and/or from the mixing chamber 330 into thesecond chamber 320. In embodiments that include the external pump 430,the external pump 430 may be configured to pump the output material 102from the second chamber 320 at a rate at least as high as a rate atwhich the pump 420 pumps the output material 102 into the output port3010.

The second chamber 320 is in communication with the open second endportion 334 of the mixing chamber 330 and the output material 102 insidethe mixing chamber 330 may flow freely from the open second end portion334 into the second chamber 320. In this manner, the output material 102does not negotiate any corners or bends between the mixing chamber 330and the second chamber 320. In the embodiment depicted, the secondchamber 320 is in communication with the entire open second end portion334 of the mixing chamber 330. The second chamber 320 may be filledcompletely with the output material 102.

The pump 420 is powered by the portion 3020 of the drive shaft 500extending through the second chamber 320. The pump 420 may besubstantially identical to the pump 410. Any pump described above assuitable for use as the pump 410 may be used for the pump 420. While thepump 410 pumps the first material 110 into the mixing chamber 330, thepump 420 pumps the output material 102 from the mixing chamber 330.Therefore, both the pump 410 and the pump 420 may be oriented to pump inthe same direction.

As is appreciated by those of ordinary skill in the art, the firstmaterial 110 may differ from the output material 102. For example, oneof the first material 110 and the output material 102 may be moreviscous than the other. Therefore, the pump 410 may differ from the pump420. The pump 410 may be configured to accommodate the properties of thefirst material 110 and the pump 420 may be configured to accommodate theproperties of the output material 102.

The pump 420 depicted in FIGS. 6 and 7, is generally referred to as asingle screw pump. In this embodiment, the pump member 4022 includes acollar portion 4030 disposed around the portion 3020 of the drive shaft500. The collar portion 4030 rotates with the portion 3020 of the driveshaft 500 as a unit. The collar portion 4030 includes one or more fluiddisplacement members 4040. The collar portion 4030 includes a singlefluid displacement member 4040 having a helical shape that circumscribesthe collar portion 4030 along a helical path.

Referring to FIG. 11, the inside of the second chamber 320 isillustrated. The pump 420 imparts an axial flow (identified by arrow“A3” and arrow “A4”) in the output material 102 inside the secondchamber 320 away from the open second end portion 334 of the mixingchamber 330.

The pump 420 may be configured to impart a circumferential flow(identified by arrow “C4”) in the output material 102 as it travels awayfrom the open second end portion 334 of the mixing chamber 330. Thecircumferential flow imparted in the output material 102 may help reducean amount of work required by the rotor 600. The circumferential flowalso directs the output material 102 toward the output port 3010.

In an alternate embodiment, the pump 420 may have substantially the sameconfiguration of the pump 410 depicted in FIG. 10. In such anembodiment, the one or more vanes 2042 are configured to impart acircumferential flow in the output material 102 as it travels away fromthe open second end portion 334 of the mixing chamber 330.

As is apparent to those of ordinary skill, various parameters of themixing device 100 may be modified to obtain different mixingcharacteristics. Exemplary parameters that may be modified include thesize of the through-holes 608, the shape of the through-holes 608, thearrangement of the through-holes 608, the number of through-holes 608,the size of the apertures 708, the shape of the apertures 708, thearrangement of the apertures 708, the number of apertures 708, the shapeof the rotor 600, the shape of the stator 700, the width of the mixingchamber 330, the length of the mixing chamber 330, rotational speed ofthe drive shaft 500, the axial velocity imparted by the internal pump410, the circumferential velocity imparted by the internal pump 410, theaxial velocity imparted by the internal pump 420, the circumferentialvelocity imparted by the internal pump 420, the configuration ofdisturbances (e.g., texture, projections, recesses, apertures, and thelike) formed on the outside surface 606 of the rotor 600, theconfiguration of disturbances (e.g., texture, projections, recesses,apertures, and the like) formed on the inside surface 706 of the stator700, and the like.

Alternate Embodiment

Referring to FIG. 12, a mixing device 5000 is depicted. The mixingdevice 5000 is an alternate embodiment of the mixing device 100.Identical reference numerals have been used herein to identifycomponents of the mixing device 5000 that are substantially similarcorresponding components of the mixing device 100. Only components ofthe mixing device 5000 that differ from the components of the mixingdevice 100 will be described.

The mixing device 5000 includes a housing 5500 for housing the rotor 600and the stator 5700. The stator 5700 may be non-rotatably coupled by itsfirst end portion 5712 and its second end portion 5714 to the housing5500. A chamber 5800 is defined between the housing 5500 and a portion5820 of the stator 5700 flanked by the first end portion 5712 and thesecond end portion 5714. The housing 5500 includes an input port 5830which provides access into the chamber 5800. The input port 5830 may beoriented substantially orthogonally to the axis of rotation “α.”however, this is not a requirement.

The stator 5700 includes a plurality of through-holes 5708 that connectthe chamber 5800 and the mixing chamber 330 (defined between the rotor600 and the stator 5700). An external pump 230 may be used to pump thethird material 130 (which may be identical to the second material 120)into the chamber 5800 via the input port 5830. The third material 130pumped into the chamber 5800 may enter the mixing chamber 330 via thethrough-holes 5708 formed in the stator 5700. The third material 130 mayforced from the channel 5800 by the pump 230, buoyancy of the thirdmaterial 130 relative to the first material 110, and a combinationthereof. As the rotor 600 rotates, it may also draw the third material130 from the channel 5800 into the mixing chamber 330. The thirdmaterial 130 may enter the mixing chamber 330 as bubbles, droplets,particles, and the like, which are imparted with a circumferentialvelocity by the rotor 600.

Alternate Embodiment

An alternate embodiment of the mixing device 100 may be constructedusing a central section 5900 depicted in FIG. 13 and a bearing housing5920 depicted in FIG. 14. FIG. 13 depicts the central section 5900having in its interior the stator 700 (see FIG. 7). Identical referencenumerals have been used herein to identify components associated withthe central section 5900 that are substantially similar correspondingcomponents of the mixing device 100. Only components of the centralsection 5900 that differ from the components of the central section 522will be described. The central section 5900 and the stator 700 are bothconstructed from a conductive material such as a metal (e.g., stainlesssteel). The input port 1010 and the output port 3010 are bothconstructed from a nonconductive material such as plastic (e.g., PET,Teflon, nylon, PVC, polycarbonate, ABS, Delrin, polysulfone, etc.).

An electrical contact 5910 is coupled to the central section 5900 andconfigured to deliver a charge thereto. The central section 5900conducts an electrical charge applied to the electrical contact 5910 tothe stator 700. In further embodiments, the central section 5900 may beconstructed from a nonconductive material. In such embodiments, theelectrical contact 5910 may pass through the central section 5900 andcoupled to the stator 700. The electric charge applied by the electricalcontact 5910 to the stator 700 may help facilitate redox or otherchemical reactions inside the mixing chamber 330.

Optionally, insulation (not shown) may be disposed around the centralsection 5900 to electrically isolate it from the environment. Further,insulation may be used between the central section 5900 and the firstand second mechanical seals 524 and 526 that flank it to isolate itelectrically from the other components of the mixing device.

Turning now to FIG. 14, the bearing housing 5920 will be described. Thebearing housing 5920 is disposed circumferentially around the portion726 of the drive shaft 500. An electrical contact 5922 is coupled to thebearing housing 5920. A rotating brush contact 5924 provides anelectrical connection between the drive shaft 500 and the electricalcontact 5922.

In this embodiment, the drive shaft 500 and the rotor 600 are bothconstructed from a conductive material such as a metal (e.g., stainlesssteel). The bearing housing 5920 may be constructed from either aconductive or a nonconductive material. An electrical charge is appliedto the drive shaft 500 by the electrical contact 5922 and the rotatingbrush contact 5924. The electrical charge is conducted by the driveshaft 500 to the rotor 600.

The alternate embodiment of the mixing device 100 constructed using thecentral section 5900 depicted in FIG. 13 and the bearing housing 5920depicted in FIG. 14 may be operated in at least two ways. First, theelectrical contacts 5910 and 5922 may be configured not to provide anelectrical charge to the stator 700 and the rotor 600, respectively. Inother words, neither of the electrical contacts 5910 and 5922 areconnected to a current source, a voltage source, and the like.

Alternatively, the electrical contacts 5910 and 5922 may be configuredto provide an electrical charge to the stator 700 and the rotor 600,respectively. For example, the electrical contacts 5910 and 5922 may becoupled to a DC voltage source (not shown) supplying a steady orconstant voltage across the electrical contacts 5910 and 5922. Thenegative terminal of the DC voltage source may be coupled to either ofthe electrical contacts 5910 and 5922 and the positive terminal of theDC voltage source may be coupled to the other of the electrical contacts5910 and 5922. The voltage supplied across the electrical contacts 5910and 5922 may range from about 0.0001 volts to about 1000 volts. Inparticular embodiments, the voltage may range from about 1.8 volts toabout 2.7 volts. By way of another example, a pulsed DC voltage having aduty cycle of between about 1% to about 99% may be used.

While the above examples of methods of operating the mixing device applya DC voltage across the electrical contacts 5910 and 5922, as isapparent to those of ordinary skill in the art, a symmetrical AC voltageor non symmetrical AC voltage having various shapes and magnitudes maybe applied across the electrical contacts 5910 and 5922 and suchembodiments are within the scope of the present invention.

Mixing Inside the Mixing Chamber 330

As mentioned above, in the prior art device 10 (shown in FIG. 1), thefirst material 110 entered the channel 32 between the rotor 12 and thestator 30 via a single limited input port 37 located along only aportion of the open second end of the channel 32. Likewise, the outputmaterial 102 exited the channel 32 via a single limited output port 40located along only a portion of the open first end of the channel 32.This arrangement caused undesirable and unnecessary friction. Byreplacing the single limited inlet port 37 and the single limited outletport 40 with the chambers 310 and 320, respectively, friction has beenreduced. Moreover, the first material 110 does not negotiate a cornerbefore entering the mixing chamber 330 and the output material 102 doesnot negotiate a corner before exiting the mixing chamber 330. Further,the chambers 310 and 320 provide for circumferential velocity of thematerial prior to entering, and after exiting the channel 32.

Accordingly, pressure drop across the mixing device 100 has beensubstantially reduced. In the embodiments depicted in FIGS. 2, 4-9, and11, the pressure drop between the input port 1010 and the output port3010 is only approximately 12 psi when the mixing device 100 isconfigured to produce about 60 gallons of the output material 102 perminute. This is an improvement over the prior art device 10 depicted inFIG. 1, which when producing about 60 gallons of output material perminute was at least 26 psi. In other words, the pressure drop across themixing device 100 is less than half that experienced by the prior artdevice 10.

According to additional aspects, the inclusion of pumps 410 and 420,which are powered by the drive shaft 500, provides a configuration thatis substantially more efficient in mixing materials and that requiresless energy than the external pumps used in the prior art.

Micro-Cavitation

During operation of the mixing device 100, the input materials mayinclude the first material 110 (e.g., a fluid) and the second material120 (e.g., a gas). The first material 110 and the second material 120are mixed inside the mixing chamber 330 formed between the rotor 600 andthe stator 700. Rotation of the rotor 600 inside the stator 700 agitatesthe first material 110 and the second material 120 inside the mixingchamber 330. The through-holes 608 formed in the rotor 600 and/or theapertures 708 formed in the stator 700 impart turbulence in the flow ofthe first material 110 and the second material 120 inside the mixingchamber 330.

Without being limited by theory, the efficiency and persistence of thediffusion of the second material 120 into the first material 110 isbelieved to be caused in part by micro-cavitation, which is described inconnection with FIGS. 15-17. Whenever a material flows over a smoothsurface, a rather laminar flow is established with a thin boundary layerthat is stationary or moving very slowly because of the surface tensionbetween the moving fluid and the stationary surface. The through-holes608 and optionally, the apertures 708, disrupt the laminar flow and cancause localized compression and decompression of the first material 110.If the pressure during the decompression cycle is low enough, voids(cavitation bubbles) will form in the material. The cavitation bubblesgenerate a rotary flow pattern 5990, like a tornado, because thelocalized area of low pressure draws the host material and the infusionmaterial, as shown in FIG. 15. When the cavitation bubbles implode,extremely high pressures result. As two aligned openings (e.g., one ofthe apertures 708 and one of the through-holes 608) pass one another, asuccussion (shock wave) occurs, generating significant energy. Theenergy associated with cavitation and succussion mixes the firstmaterial 110 and the second material 120 together to an extremely highdegree, perhaps at the molecular level.

The tangential velocity of the rotor 600 and the number of openings thatpass each other per rotation may dictate the frequency at which themixing device 100. It has been determined that operating the mixingdevice 100 within in the ultrasonic frequency range can be beneficial inmany applications. It is believed that operating the mixing device 100in the ultrasonic region of frequencies provides the maximum successionshock energy to shift the bonding angle of the fluid molecule, whichenables it to transport an additional quantity of the second material120 which it would not normally be able to retain. When the mixingdevice 100 is used as a diffuser, the frequency at which the mixingdevice 100 operates appears to affect the degree of diffusion, leadingto much longer persistence of the second material 120 (infusionmaterial) in the first material 110 (host material).

Referring now to FIG. 15, an alternate embodiment of the rotor 600,rotor 6000 is provided. The cavitations created within the firstmaterial 110 in the mixing chamber 330 may be configured to occur atdifferent frequencies along the length of the mixing chamber 330. Thefrequencies of the cavitations may be altered by altering the numberand/or the placement of the through-holes 6608 along the length of therotor 600. Each of the through-holes 6608 may be substantially similarto the through-holes 608 (discussed above).

By way of non-limiting example, the rotor 6000 may be subdivided intothree separate exemplary sections 6100, 6200, and 6300. Thethrough-holes 6608 increase in density from the section 6100 to thesection 6200, the number of holes in the section 6100 being greater thanthe number of holes in the section 6200. The through-holes 6608 alsoincrease in density from the section 6200 to the section 6300, thenumber of holes in the section 6200 being greater than the number ofholes in the section 6300. Each of the sections 6100, 6200, and 6300create succussions within their particular area at a different frequencydue to the differing numbers of through-holes 6608 formed therein.

By manufacturing the rotor 6000 with a desired number of through-holes6608 appropriately arranged in a particular area, the desired frequencyof the succussions within the mixing chamber 330 may be determined.Similarly, the desired frequency of the cavitations may be determined bya desired number of apertures 708 appropriately arranged in a particulararea upon the stator 700 within which the rotor 600 rotates. Further,the desired frequency (or frequencies) of the succussions within themixing chamber 330 may be achieved by selecting both a particular numberand arrangement of the apertures 708 formed in the stator 700 and aparticular number and arrangement of the through-holes 608 formed in therotor 600.

FIGS. 19-21, depict various alternative arrangements of the apertures708 formed in the stator 700 and the through-holes 608 formed in therotor 600 configured to achieve different results with respect to thecavitations created. FIG. 19 illustrates a configuration in which theapertures 708 and the through-holes 608 are aligned along an axis 7000that is not parallel with any line (e.g., line 7010) drawn through theaxis of rotation “α” of the rotor 600. In other words, if the rotor 600has a cylindrical shape, the axis 7000 does not pass through the centerof the rotor 600. Thus, the first material 110 within the mixing chamber330 will not be oriented perpendicularly to the compressions anddecompressions created by the apertures 708 and the through-holes 608.The compressions and decompressions will instead have a force vectorthat has at least a component parallel to the circumferential flow (inthe direction of arrow “C3” of FIG. 9) of first material 110 within themixing chamber 330.

Relative alignment of the apertures 708 and the through-holes 608 mayalso affect the creation of cavitations in the mixing chamber 330. FIG.20 illustrates an embodiment in which the apertures 708 are inregistration across the mixing chamber 330 with the through-holes 608.In this embodiment, rotation of the rotor 600 brings the through-holes608 of the rotor into direct alignment with the apertures 708 of thestator 700. When in direct alignment with each other, the compressiveand decompressive forces created by the apertures 708 and thethrough-holes 608 are directly aligned with one another.

In the embodiment depicted in FIG. 21, the apertures 708 and thethrough-holes 608 are offset by an offset amount “X” along the axis ofrotation “α.”. By way of non-limiting example, the offset amount “X” maybe determined as a function of the size of the apertures 708. Forexample, the offset amount “X” may be approximately equal to one half ofthe diameter of the apertures 708. Alternatively, the offset amount “X”may be determined as a function of the size of the through-holes 608.For example, the offset amount “X” may be approximately equal to onehalf of the diameter of the through-holes 608. If features (e.g.,recesses, projections, etc.) other than or in addition to thethrough-holes 608 and the apertures 708 are included in either the rotor600 or the stator 700, the offset amount “X” may be determined as afunction of the size of such features. In this manner, the compressiveand decompressive forces caused by the apertures 708 of the stator 700and the through-holes 608 of the rotor 600 collide at a slight offsetcausing additional rotational and torsional forces within the mixingchamber 330. These additional forces increase the mixing (e.g.,diffusive action) of the second material 120 into the first material 110within the mixing chamber 330.

Referring now to FIGS. 22-25, non-limiting examples of suitablecross-sectional shapes for the apertures 708 and the through-holes 608are provided. The cross-sectional shape of the apertures 708 and/or thethrough-holes 608 may be square as illustrated in FIG. 22, circular asillustrated in FIG. 23, and the like.

Various cross-sectional shapes of apertures 708 and/or the through-holes608 may be used to alter flow of the first material 110 as the rotor 600rotates within the stator 700. For example, FIG. 24 depicts a teardropcross-sectional shape having a narrow portion 7020 opposite a wideportion 7022. If the through-holes 608 have this teardrop shape, whenthe rotor 600 is rotated (in the direction generally indicated by thearrow “F”), the forces exerted on the first material 110, the secondmaterial 120, and optionally the third material 130 within the mixingchamber 330 increase as the materials pass from the wide portion 7022 ofthe teardrop to the narrow portion 7020.

Additional rotational forces can be introduced into the mixing chamber330 by forming the apertures 708 and/or the through-holes 608 with aspiral configuration as illustrated in FIG. 25. Material that flows intoand out of the apertures 708 and/or the through-holes 608 having thespiral configuration experience a rotational force induced by the spiralconfiguration. The examples illustrated in FIGS. 22-25 are provided asnon-limiting illustrations of alternate embodiments that may be employedwithin the mixing device 100. By application of ordinary skill in theart, the apertures 708 and/or the through-holes 608 may be configured innumerous ways to achieve various succussive and agitative forcesappropriate for mixing materials within the mixing chamber 330.

Double Layer Effect

The mixing device 100 may be configured to create the output material102 by complex and non-linear fluid dynamic interaction of the firstmaterial 110 and the second material 120 with complex, dynamicturbulence providing complex mixing that further favors electrokineticeffects (described below). The result of these electrokinetic effectsmay be observed within the output material 102 as charge redistributionsand redox reactions, including in the form of solvated electrons thatare stabilized within the output material.

Ionization or dissociation of surface groups and/or adsorption of ionsfrom a liquid cause most solid surfaces in contact with the liquid tobecome charged. Referring to FIG. 26, an electrical double layer (“EDL”)7100 forms around exemplary surface 7110 in contact with a liquid 7120.In the EDL 7100, ions 7122 of one charge (in this case, negativelycharged ions) adsorb to the surface 7120 and form a surface layer 7124typically referred to as a Stern layer. The surface layer 7124 attractscounterions 7126 (in this case, positively charged ions) of the oppositecharge and equal magnitude, which form a counterion layer 7128 below thesurface layer 7124 typically referred to as a diffuse layer. Thecounterion layer 7128 is more diffusely distributed than the surfacelayer 7124 and sits upon a uniform and equal distribution of both ionsin the bulk material 7130 below. For OH— and H+ ions in neutral water,the Gouy-Chapman model would suggest that the diffuse counterion layerextends about one micron into the water.

According to particular aspects, the electrokinetic effects mentionedabove are caused by the movement of the liquid 7120 next to the chargedsurface 7110. Within the liquid 7120 (e.g., water, saline solution, andthe like), the adsorbed ions 7122 forming the surface layer 7124 arefixed to the surface 7120 even when the liquid 7120 is in motion (forexample, flowing in the direction indicated by arrow “G”); however, ashearing plane 7132 exists within the diffuse counterion layer 7128spaced from the surface 7120. Thus, as the liquid 7120 moves, some ofthe diffuse counterions 7126 are transported away from the surface 7120,while the absorbed ions 7122 remain at the surface 7120. This produces aso-called ‘streaming current.’

Within the mixing chamber 330, the first material 110, the secondmaterial 120, and optionally, the third material 130 are subject to anelectromagnetic field created by the inside surface 705 of the stator700 and/or the outside surface 606 of the rotor 600, a voltage betweenthe inside surface 705 and the outside surface 606, and/or anelectrokinetic effect (e.g., streaming current) caused by at least oneEDL formed in the first material 110. The at least one EDL may beintroduced into the first material 110 by at least one of the insidesurface 705 of the stator 700 and the outside surface 606 of the rotor600.

Movement of the first material 110 through the mixing chamber 330relative to surface disturbances (e.g., the through-holes 608 andapertures 708) creates cavitations in the first material 110 within themixing chamber 330, which may diffuse the second material 120 into thefirst material 110. These cavitations may enhance contact between of thefirst material 110 and/or the second material 120 with the electricdouble layer formed on the inside surface 705 of the stator 700 and/orthe electric double layer formed on the outside surface 606 of the rotor600. Larger surface to volume ratios of the mixing chamber, an increaseddwell time of the combined materials within the mixing chamber, andfurther in combination with a smaller average bubble size (and hencesubstantially greater bubble surface area) provide for effectivelyimparting EDL-mediated effects to the inventive output materials.

In embodiments in which the inside surface 705 and the outside surface606 are constructed from a metallic material, such as stainless steel,the motion of the liquid 7120 and/or the streaming current(s) facilitateredox reactions involving H₂O, OH—, H+, and O₂ at the inside surface 705and the outside surface 606.

Referring to FIG. 27, without being limited by theory, it is believed asection 7140 of the mixing chamber 330 between the inside surface 705and the outside surface 606 may be modeled as a pair of parallel plates7142 and 7144. If the first material 110 is a liquid, the first material110 enters the section 7140 through an inlet “IN” and exits the section7140 through an outlet “OUT.” The inlet “IN” and the outlet “OUT”restrict the flow into and out of the section 7140.

Referring to FIG. 28, the area between the parallel plates 7142 and 7144has a high surface area to volume ratio. Hence, a substantial portion ofthe counterion layer 7128 (and counterions 7126) may be in motion as thefirst material 110 moves between the plates 7142 and 7144. The number ofcounterions 7126 in motion may exceed the number allowed to enter thesection 7140 by the inlet “IN” and the number allowed to exit thesection 7140 by the outlet “OUT.” The inlet “IN” and the outlet “OUT”feeding and removing the first material 110 from the section 7140,respectively, have far less surface area (and a lower surface area tovolume ratio) than the parallel plates 7142 and 7144 and thereby reducethe portion of the counterions 7126 in motion in the first material 110entering and leaving the section 7140. Therefore, entry and exit fromthe section 7140 increases the streaming current locally. While abackground streaming current (identified by arrow “BSC”) caused by theflowing first material 110 over any surface is always present inside themixing device 100, the plates 7142 and 7144 introduce an increased“excess” streaming current (identified by arrow “ESC”) within thesection 7140.

Without a conductive return current (identified by arrow “RC”) in theplates 7142 and 7144 in the opposite direction of the flow of the firstmaterial 110, an excess charge 7146 having the same sign as theadsorbing ions 7122 would accumulate near the inlet “IN,” and an excesscharge 7148 having the same sign as the counterion 7126 would accumulatenear the at outlet “OUT.” Because such accumulated charges 7146 and7148, being opposite and therefore attracted to one another, cannotbuild up indefinitely the accumulated charges seek to join together byconductive means. If the plates 7142 and 7144 are perfectly electricallyinsulating, the accumulated charges 7146 and 7148 can relocate onlythrough the first material 110 itself. When the conductive returncurrent (identified by arrow “RC”) is substantially equivalent to theexcess streaming current (identified by arrow “ESC”) in the section7140, a steady-state is achieved having zero net excess streamingcurrent, and an electrostatic potential difference between the excesscharge 7146 near the inlet “IN,” and the excess charge 7148 near theoutlet “OUT” creating a steady-state charge separation therebetween.

The amount of charge separation, and hence the electrostatic potentialdifference between the excess charge 7146 near the inlet “IN,” and theexcess charge 7148 near the outlet “OUT,” depends on additional energyper unit charge supplied by a pump (e.g., the rotor 600, the internalpump 410, and/or the external pump 210) to “push” charge against theopposing electric field (created by the charge separation) to produce aliquid flow rate approximating a flow rate obtainable by a liquidwithout ions (i.e., ions 7122 and 7126). If the plates 7142 and 7144 areinsulators, the electrostatic potential difference is a direct measureof the EMF the pump (e.g., the rotor 600, the internal pump 410 and/orthe external pump 210) can generate. In this case, one could measure theelectrostatic potential difference using a voltmeter having a pair ofleads by placing one of the leads in the first material 110 near theinlet “IN,” and the other lead in the first material 110 near the outlet“OUT.”

With insulating plates 7142 and 7144, any return current is purely anion current (or flow of ions), in that the return current involves onlythe conduction of ions through the first material 110. If otherconductive mechanisms through more conductive pathways are presentbetween the excess charge 7146 near the inlet “IN,” and the excesscharge 7148 near the outlet “OUT,” the return current may use those moreconductive pathways. For example, conducting metal plates 7142 and 7144may provide more conductive pathways; however, these more conductivepathways transmit only an electron current and not the ion current.

As is appreciated by those of ordinary skill, to transfer the chargecarried by an ion to one or more electrons in the metal, and vise versa,one or more oxidation-reduction reactions must occur at the surface ofthe metal, producing reaction products. Assuming the first material 110is water (H₂O) and the second material 120 is oxygen (O₂), anon-limiting example of a redox reaction, which would inject negativecharge into the conducting plates 7142 and 7144 includes the followingknown half-cell reaction:O₂+H₂O→O₃+2H⁺+2e ⁻,

Again, assuming the first material 110 is water (H₂O) and the secondmaterial 120 is oxygen (O₂), a non-limiting example of a redox reactionincludes the following known half-cell reaction, which would removenegative charge from the conducting plates 7142 and 7144 includes thefollowing known half-cell reaction:2H⁺ +e ⁻→H₂,

With conducting metal plates 7142 and 7144, most of the return currentis believed to be an electron current, because the conducting plates7142 and 7144 are more conductive than the first material 110 (providedthe redox reactions are fast enough not to be a limiting factor). Forthe conducting metal plates 7142 and 7144, a smaller charge separationaccumulates between the inlet “IN” and the outlet “OUT,” and a muchsmaller electrostatic potential exists therebetween. However, this doesnot mean that the EMF is smaller.

As described above, the EMF is related to the energy per unit charge thepump provides to facilitate the flow of the first material 110 againstthe opposing electric field created by the charge separation. Becausethe electrostatic potential is smaller, the pump may supply less energyper unit charge to cause the first material 110 to flow. However, theabove example redox reactions do not necessarily occur spontaneously,and thus may require a work input, which may be provided by the pump.Therefore, a portion of the EMF (that is not reflected in the smallerelectrostatic potential difference) may be used to provide the energynecessary to drive the redox reactions.

In other words, the same pressure differentials provided by the pump topush against the opposing electric field created by the chargeseparation for the insulating plates 7142 and 7144, may be used both to“push” the charge through the conducting plates 7142 and 7144 and drivethe redox reactions.

Referring to FIG. 29, an experimental setup for an experiment conductedby the inventors is provided. The experiment included a pair ofsubstantially identical spaced apart 500 ml standard Erlenmeyer flasks7150 and 7152, each containing a volume of deionized water 7153. Arubber stopper 7154 was inserted in the open end of each of the flasks7150 and 7152. The stopper 7154 included three pathways, one each for ahollow tube 7156, a positive electrode 7158, and a negative electrode7160. With respect to each of the flasks 7150 and 7152, each of thehollow tube 7156, the positive electrode 7158, and the negativeelectrode 7160 all extended from outside the flask, through the stopper7154, and into the deionized water 7153 inside the flask. The positiveelectrode 7158 and the negative electrode 7160 were constructed fromstainless steel. The hollow tubes 7156 in both of the flasks 7150 and7152 had an open end portion 7162 coupled to a common oxygen supply7164. The positive electrode 7158 and the negative electrode 7160inserted into the flask 7152 where coupled to a positive terminal and anegative terminal, respectively, of a DC power supply 7168. Exactly thesame sparger was used in each flask.

Oxygen flowed through the hollow tubes 7156 into both of the flasks 7150and 7152 at a flow rate (Feed) of about 1 SCFH to about 1.3 SCFH(combined flow rate). The voltage applied across the positive electrode7158 and the negative electrode 7160 inserted into the flask 7152 wasabout 2.55 volts. This value was chosen because it is believed to be anelectrochemical voltage value sufficient to affect all oxygen species.This voltage was applied continuously over three to four hours duringwhich oxygen from the supply 7164 was bubbled into the deionized water7153 in each of the flasks 7150 and 7152.

Testing of the deionized water 7153 in the flask 7150 with HRP andpyrogallol gave an HRP-mediated pyrogallol reaction activity, consistentwith the properties of fluids produced with the alternate rotor/statorembodiments described herein. The HRP optical density was about 20%higher relative to pressure-pot or fine-bubbled solutions of equivalentoxygen content. The results of this experiment indicate that mixinginside the mixing chamber 330 involves a redox reaction. According toparticular aspects, the inventive mixing chambers provide for outputmaterials comprising added electrons that are stabilized by eitheroxygen-rich water structure within the inventive output solutions, or bysome form of oxygen species present due to the electrical effects withinthe process.

Additionally, the deionized water 7153 in both of the flasks 7150 and7152 was tested for both ozone and hydrogen peroxide employing industrystandard calorimetric test ampules with a sensitivity of 0.1 ppm forhydrogen peroxide and 0.6 ppm for ozone. There was no positiveindication of either species up to the detection limits of thoseampules.

Dwell Time

Dwell time is an amount of time the first material 110, the secondmaterial 120, and optionally the third material 130 spend in the mixingchamber 330. The ratio of the length of the mixing chamber 330 to thediameter of the mixing chamber 330 may significantly affect dwell time.The greater the ratio, the longer the dwell time. As mentioned in theBackground Section, the rotor 12 of the prior art device 10 (see FIG. 1)had a diameter of about 7.500 inches and a length of about 6.000 inchesproviding a length to diameter ratio of about 0.8. In contrast, inparticular embodiments, the length of the mixing chamber 330 of themixing device 100 is about 5 inches and the diameter “D1” of the rotor600 is about 1.69 inches yielding a length to diameter ratio of about2.95.

Dwell time represents the amount of time that the first material 110,the second material 120, and optionally the third material 130 are ableto interact with the electrokinetic phenomena described herein. Theprior art device 10 is configured to produce about 60 gallons of theoutput material 102 per minute and the mixing device 100 is configuredto produce about 0.5 gallons of the output material 102 per minute, theprior art device 10 (see FIG. 1) had a fluid dwell time of about 0.05seconds, whereas embodiments of the mixing device 100 have asubstantially greater (about 7-times greater) dwell time of about 0.35seconds. This longer dwell time allows the first material 110, thesecond material 120, and optionally the third material 130 to interactwith each other and the surfaces 606 and 705 (see FIG. 7) inside themixing chamber 330 for about 7 times longer than was possible in theprior art device 10. In additional embodiments, the dwell time is atleast 1.5-times, at least 2-times, at least 3-times, at least 4-times,at least 5-times, at least 6-times, at least 7-times or greater, thanwas possible in the prior art device 10.

With reference to Table 3I below, the above dwell times were calculatedby first determining the flow rate for each device in gallons persecond. In the case of the prior art device 10 was configured to operateat about 60 gallons of output material per minute, while the mixingdevice 100 is configured to operate over a broader range of flow rate,including at an optimal range of about 0.5 gallons of output materialper minute. The flow rate was then converted to cubic inches per secondby multiplying the flow rate in gallons per second by the number ofcubic inches in a gallon (i.e., 231 cubic inches). Then, the volume(12.876 cubic inches) of the channel 32 of the prior art device 10 wasdivided by the flow rate of the device (231 cubic inches/second) toobtain the dwell time (in seconds) and the volume (0.673 cubic inches)of the mixing chamber 330 of the mixing device 100 was divided by theflow rate (1.925 cubic inches/second) of the device (in cubic inches persecond) to obtain the dwell time (in seconds).

TABLE 3 Inventive device can accommodate a range of dwell times,including a substantially increased (e.g., 7-times) dwell time relativeto prior art devices. Flow Flow Rate Volume Rate Flow Rate Cubic MixingDwell Gallons/ Gallons/ Inches/ Chamber Time Device Minute Second Second(Cubic Inches) (Seconds) Prior art 60 1.000 231.000 12.876 0.056 device10 Mixing 2 0.033 7.700 0.673 0.087 device 100 Mixing 0.5 0.008 1.9250.673 0.350 device 100

Rate of Infusion

Particular aspects of the mixing device 100 provide an improved oxygeninfusion rate over the prior art, including over prior art device 10(see FIG. 1). When the first material 110 is water and the secondmaterial 120 is oxygen, both of which are processed by the mixing device100 in a single pass (i.e., the return block of FIG. 2 is set to “NO”)at or near 20° Celsius, the output material 102 has a dissolved oxygenlevel of about 43.8 parts per million. In certain aspects, an outputmaterial having about 43.8 ppm dissolved oxygen is created in about 350milliseconds via the inventive flow through the inventive nonpressurized (non-pressure pot) methods. In contrast, when the firstmaterial 110 (water) and the second material 120 (oxygen) are bothprocessed in a single pass at or near 20° Celsius by the prior artdevice 10, the output material had dissolved oxygen level of only 35parts per million in a single pass of 56 milliseconds.

Output Material 102

When the first material 110 is a liquid (e.g., freshwater, saline,GATORADE®, and the like) and the second material 120 is a gas (e.g.,oxygen, nitrogen, and the like), the mixing device 100 may diffuse thesecond material 120 into the first material 110. The following discussesresults of analyses performed on the output material 102 to characterizeone or more properties of the output material 102 derived from havingbeen processed by the mixing device 100.

When the first material 110 is saline solution and the second material120 is oxygen gas, experiments have indicated that a vast majority ofoxygen bubbles produced within the saline solution are no greater than0.1 micron in size.

Decay of Dissolved Oxygen Levels

Referring now to FIG. 30, there is illustrated the DO levels in waterprocessed with oxygen in the mixing device 100 and stored in a 500 mlthin-walled plastic bottle and a 1000 ml glass bottle. Each of thebottles was capped and stored at 65° Fahrenheit. Point 7900 is the DOlevel at bottling. Line 7902 illustrates the Henry's Law equilibriumstate (i.e., the amount of dissolved oxygen that should be within thewater at 65° Fahrenheit), which is a DO level of slightly less than 10ppm. Points 7904 and 7906 represent the DO levels within the water inthe plastic bottle at 65 days and 95 days respectively. As can be seenat point 7904, when the plastic bottle is opened approximately 65 daysafter bottling, the DO level within the water is approximately 27.5 ppm.When the bottle is opened approximately 95 days after bottling, asindicated at point 7906, the DO level is approximately 25 ppm. Likewise,for the glass bottle, the DO level is approximately 40 ppm at 65 days asindicated at point 7908 and is approximately 41 ppm at 95 days asillustrated at point 7910. Thus, FIG. 30 indicates the DO levels withinboth the plastic bottle and the glass bottle remain relatively high at65° Fahrenheit.

Referring to FIG. 31, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlplastic thin-walled bottle and a 1000 ml glass bottle. Both bottles wererefrigerated at 39 degrees Fahrenheit. Again, DO levels of theoxygen-enriched fluid remained steady and decreased only slightly out toat least 365 days.

Molecular Interactions

Conventionally, quantum properties are thought to belong to elementaryparticles of less than 10⁻¹⁰ meters, while the macroscopic world of oureveryday life is referred to as classical, in that it behaves accordingto Newton's laws of motion.

Recently, molecules have been described as forming clusters thatincrease in size with dilution. These clusters measure severalmicrometers in diameter, and have been reported to increase in sizenon-linearly with dilution. Quantum coherent domains measuring 100nanometers in diameter have been postulated to arise in pure water, andcollective vibrations of water molecules in the coherent domain mayeventually become phase locked to electromagnetic field fluctuations,providing for stable oscillations in water, providing a form of ‘memory’in the form of excitation of long lasting coherent oscillations specificto dissolved substances in the water that change the collectivestructure of the water, which may in turn determine the specificcoherent oscillations that develop. Where these oscillations becomestabilized by magnetic field phase coupling, the water, upon diluctionmay still carry ‘seed’ coherent oscillations. As a cluster of moleculesincreases in size, its electromagnetic signature is correspondinglyamplified, reinforcing the coherent oscillations carried by the water.

Despite variations in the cluster size of dissolved molecules anddetailed microscopic structure of the water, a specificity of coherentoscillations may nonetheless exist. One model for considering changes inproperties of water is based on considerations involved incrystallization.

With reference to FIG. 36, a simplified protonated water cluster forminga nanoscale cage 8700 is shown. A protonated water cluster typicallytakes the form of H⁺(H₂0)_(n). Some protonated water clusters occurnaturally, such as in the ionosphere. Without being bound by anyparticular theory, and according to particular aspects, other types ofwater clusters or structures (clusters, nanocages, etc) are possible,including structures comprising oxygen and stabilized electrons impartedto the inventive output materials. Oxygen atoms 8704 may be caught inthe resulting structures 8700. The chemistry of the semi-bound nanocageallows the oxygen 8704 and/or stabilized electrons to remain dissolvedfor extended periods of time. Other atoms or molecules, such asmedicinal compounds, can be caged for sustained delivery purposes. Thespecific chemistry of the solution material and dissolved compoundsdepend on the interactions of those materials.

Fluids processed by the mixing device 100 have been shown viaexperiments to exhibit different structural characteristics that areconsistent with an analysis of the fluid in the context of a clusterstructure.

Water processed through the mixing device 100 has been demonstrated tohave detectible structural differences when compared with normalunprocessed water. For example, processed water has been shown to havemore Rayleigh scattering than is observed in unprocessed water. In theexperiments that were conducted, samples of processed and unprocessedwater were prepared (by sealing each in a separate bottle), coded (forlater identification of the processed sample and unprocessed sample),and sent to an independent testing laboratory for analysis. Only afterthe tests were completed were the codes interpreted to reveal whichsample had been processed by the mixing device 100.

At the laboratory, the two samples were placed in a laser beam having awavelength of 633 nanometers. The fluid had been sealed in glass bottlesfor approximately one week before testing. With respect to the processedsample, Sample B scattered light regardless of its position relative tothe laser source. However, Sample A did not. After two to three hoursfollowing the opening of the bottle, the scattering effect of Sample Bdisappeared. These results imply the water exhibited a memory causingthe water to retain its properties and dissipate over time. Theseresults also imply the structure of the processed water is opticallydifferent from the structure of the unprocessed fluid. Finally, theseresults imply the optical effect is not directly related to DO levelsbecause the DO level at the start was 45 ppm and at the end of theexperiment was estimated to be approximately 32 ppm.

Charge-Stabilized Nanostructures (e.g., Charge StabilizedOxygen-Containing Nanostructures):

As described herein above under “Double Layer Effect,” “Dwell Time,”“Rate of Infusion,” and “Bubble size Measurements,” the mixing device100 creates, in a matter of milliseconds, a unique non-linear fluiddynamic interaction of the first material 110 and the second material120 with complex, dynamic turbulence providing complex mixing in contactwith an effectively enormous surface area (including those of the deviceand of the exceptionally small gas bubbles of less that 100 nm) thatprovides for the novel electrokinetic effects described herein.Additionally, feature-localized electrokinetic effects (voltage/current)were demonstrated herein (see working Example 20) using a speciallydesigned mixing device comprising insulated rotor and stator features.

As well-recognized in the art, charge redistributions and/or solvatedelectrons are known to be highly unstable in aqueous solution. Accordingto particular aspects, Applicants' electrokinetic effects (e.g., chargeredistributions, including, in particular aspects, solvated electrons)are surprisingly stabilized within the output material (e.g., salinesolutions, ionic solutions). In fact, as described herein, the stabilityof the properties and biological activity of the inventiveelectrokinetic fluids (e.g., RNS-60 or Solas) can be maintained formonths in a gas-tight container, indicating involvement of dissolved gas(e.g., oxygen) in helping to generate and/or maintain, and/or mediatethe properties and activities of the inventive solutions. Significantly,as described in the working Examples herein, the charge redistributionsand/or solvated electrons are stably configured in the inventiveelectrokinetic ionic aqueous fluids in an amount sufficient to provide,upon contact with a living cell (e.g., mammalian cell) by the fluid,modulation of at least one of cellular membrane potential and cellularmembrane conductivity (see, e.g., cellular patch clamp working Examples23 and 24).

As described herein under “Molecular Interactions,” to account for thestability and biological compatibility of the inventive electrokineticfluids (e.g., electrokinetic saline solutions), Applicants have proposedthat interactions between the water molecules and the molecules of thesubstances (e.g., oxygen) dissolved in the water change the collectivestructure of the water and provide for nanoscale cage clusters,including nanostructures comprising oxygen and/or stabilized electronsimparted to the inventive output materials. Without being bound bymechanism, and according to the properties and activities describedherein, the configuration of the nanostructures in particular aspects issuch that they: comprise (at least for formation and/or stability and/orbiological activity) dissolved gas (e.g., oxygen); enable theelectrokinetic fluids (e.g., RNS-60 or Solas saline fluids) to modulate(e.g., impart or receive) charges and/or charge effects upon contactwith a cell membrane or related constituent thereof; and in particularaspects provide for stabilization (e.g., carrying, harboring, trapping)solvated electrons in a biologically-relevant form.

According to particular aspects, and as supported by the presentdisclosure, in ionic or saline (e.g., standard saline, NaCl) solutions,the inventive nanostructures comprise charge stabilized nanostrutures(e.g., average diameter less that 100 nm) that may comprise at least onedissolved gas molecule (e.g., oxygen) within a charge-stabilizedhydration shell. According to additional aspects, and as describedelsewhere herein, the charge-stabilized hydration shell may comprise acage or void harboring the at least one dissolved gas molecule (e.g.,oxygen). According to further aspects, by virtue of the provision ofsuitable charge-stabilized hydration shells, the charge-stabilizednanostructure and/or charge-stabilized oxygen containing nano-structuresmay additionally comprise a solvated electron (e.g., stabilized solvatedelectron).

Without being bound by mechanism or particular theory, after the presentpriority date, charge-stabilized microbubbles stabilized by ions inaqueous liquid in equilibrium with ambient (atmospheric) gas have beenproposed (Bunkin et al., Journal of Experimental and TheoreticalPhysics, 104:486-498, 2007; incorporated herein by reference in itsentirety). According to particular aspects of the present invention,Applicants' novel electrokinetic fluids comprise a novel, biologicallyactive form of charge-stabilized oxygen-containing nanostructures, andmay further comprise novel arrays, clusters or associations of suchstructures.

According to the charge-stabilized microbubble model, the short-rangemolecular order of the water structure is destroyed by the presence of agas molecule (e.g., a dissolved gas molecule initially complexed with anonadsorptive ion provides a short-range order defect), providing forcondensation of ionic droplets, wherein the defect is surrounded byfirst and second coordination spheres of water molecules, which arealternately filled by adsorptive ions (e.g., acquisition of a screeningshell of Na⁺ ions to form an electrical double layer) and nonadsorptiveions (e.g., Cl⁻ ions occupying the second coordination sphere) occupyingsix and 12 vacancies, respectively, in the coordination spheres. Inunder-saturated ionic solutions (e.g., undersaturated saline solutions),this hydrated ‘nucleus’ remains stable until the first and secondspheres are filled by six adsorptive and five nonadsorptive ions,respectively, and then undergoes Coulomb explosion creating an internalvoid containing the gas molecule, wherein the adsorptive ions (e.g., Na⁺ions) are adsorbed to the surface of the resulting void, while thenonadsorptive ions (or some portion thereof) diffuse into the solution(Bunkin et al., supra). In this model, the void in the nanostructure isprevented from collapsing by Coulombic repulsion between the ions (e.g.,Na⁺ ions) adsorbed to its surface. The stability of the void-containingnanostrutures is postulated to be due to the selective adsorption ofdissolved ions with like charges onto the void/bubble surface anddiffusive equilibrium between the dissolved gas and the gas inside thebubble, where the negative (outward electrostatic pressure exerted bythe resulting electrical double layer provides stable compensation forsurface tension, and the gas pressure inside the bubble is balanced bythe ambient pressure. According to the model, formation of suchmicrobubbles requires an ionic component, and in certain aspectscollision-mediated associations between particles may provide forformation of larger order clusters (arrays) (Id).

The charge-stabilized microbubble model suggests that the particles canbe gas microbubbles, but contemplates only spontaneous formation of suchstructures in ionic solution in equilibrium with ambient air, isuncharacterized and silent as to whether oxygen is capable of formingsuch structures, and is likewise silent as to whether solvated electronsmight be associated and/or stabilized by such structures.

According to particular aspects, the inventive electrokinetic fluidscomprising charge-stabilized nanostructures and/or charge-stabilizedoxygen-containing nanostructures are novel and fundamentally distinctfrom the postulated non-electrokinetic, atmospheric charge-stabilizedmicrobubble structures according to the microbubble model.Significantly, this conclusion is in unavoidable, deriving, at least inpart, from the fact that control saline solutions do not have thebiological properties disclosed herein, whereas Applicants'charge-stabilized nanostructures provide a novel, biologically activeform of charge-stabilized oxygen-containing nanostructures.

According to particular aspects of the present invention, Applicants'novel electrokinetic device and methods provide for novelelectrokinetically-altered fluids comprising significant quantities ofcharge-stabilized nanostructures in excess of any amount that may or maynot spontaneously occur in ionic fluids in equilibrium with air, or inany non-electrokinetically generated fluids. In particular aspects, thecharge-stabilized nanostructures comprise charge-stabilizedoxygen-containing nanostructures. In additional aspects, thecharge-stabilized nanostrutures are all, or substantially allcharge-stabilized oxygen-containing nanostructures, or thecharge-stabilized oxygen-containing nanostructures the majorcharge-stabilized gas-containing nanostructure species in theelectrokinetic fluid.

According to yet further aspects, the charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures maycomprise or harbor a solvated electron, and thereby provide a novelstabilized solvated electron carrier. In particular aspects, thecharge-stabilized nanostructures and/or the charge-stabilizedoxygen-containing nanostructures provide a novel type of electride (orinverted electride), which in contrast to conventional solute electrideshaving a single organically coordinated cation, rather have a pluralityof cations stably arrayed about a void or a void containing an oxygenatom, wherein the arrayed sodium ions are coordinated by water hydrationshells, rather than by organic molecules. According to particularaspects, a solvated electron may be accommodated by the hydration shellof water molecules, or preferably accommodated within the nanostructurevoid distributed over all the cations. In certain aspects, the inventivenanostructures provide a novel ‘super electride’ structure in solutionby not only providing for distribution/stabilization of the solvatedelectron over multiple arrayed sodium cations, but also providing forassociation or partial association of the solvated electron with thecaged oxygen molecule(s) in the void—the solvated electron distributingover an array of sodium atoms and at least one oxygen atom. According toparticular aspects, therefore, ‘solvated electrons’ as presentlydisclosed in association with the inventive electrokinetic fluids, maynot be solvated in the traditional model comprising direct hydration bywater molecules. Alternatively, in limited analogy with dried electridesalts, solvated electrons in the inventive electrokinetic fluids may bedistributed over multiple charge-stabilized nanostructures to provide a‘lattice glue’ to stabilize higher order arrays in aqueous solution.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures arecapable of interacting with cellular membranes or constituents thereof,or proteins, etc., to mediate biological activities. In particularaspects, the inventive charge-stabilized nanostructures and/or thecharge-stabilized oxygen-containing nanostructures harboring a solvatedelectron are capable of interacting with cellular membranes orconstituents thereof, or proteins, etc., to mediate biologicalactivities.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures interactwith cellular membranes or constituents thereof, or proteins, etc., as acharge and/or charge effect donor (delivery) and/or as a charge and/orcharge effect recipient to mediate biological activities. In particularaspects, the inventive charge-stabilized nanostructures and/or thecharge-stabilized oxygen-containing nanostructures harboring a solvatedelectron interact with cellular membranes as a charge and/or chargeeffect donor and/or as a charge and/or charge effect recipient tomediate biological activities.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures areconsistent with, and account for the observed stability and biologicalproperties of the inventive electrokinetic fluids, and further provide anovel electride (or inverted electride) that provides for stabilizedsolvated electrons in aqueous ionic solutions (e.g., saline solutions,NaCl, etc.).

In particular aspects, the charge-stabilized oxygen-containingnanostructures substantially comprise, take the form of, or can giverise to, charge-stabilized oxygen-containing nanobubbles. In particularaspects, charge-stabilized oxygen-containing clusters provide forformation of relatively larger arrays of charge-stabilizedoxygen-containing nanostructures, and/or charge-stabilizedoxygen-containing nanobubbles or arrays thereof. In particular aspects,the the charge-stabilized oxygen-containing nanostructures can providefor formation of hydrophobic nanobubbles upon contact with a hydrophobicsurface (see elsewhere herein under EXAMPLE 25).

In particular aspects, the charge-stabilized oxygen-containingnanostructures substantially comprise at least one oxygen molecule. Incertain aspects, the charge-stabilized oxygen-containing nanostructuressubstantially comprise at least 1, at least 2, at least 3, at least 4,at least 5, at least 10 at least 15, at least 20, at least 50, at least100, or greater oxygen molecules. In particular aspects,charge-stabilized oxygen-containing nanostructures comprise or give riseto nanobubbles (e.g., hydrophobid nanobubbles) of about 20 nm×1.5 nm,comprise about 12 oxygen molecules (e.g., based on the size of an oxygenmolecule (approx 0.3 nm by 0.4 nm), assumption of an ideal gas andapplication of n=PV/RT, where P=1 atm, R=0.082□057□l.atm/mol·K; T=295K;V=pr²h=4.7×10⁻²² L, where r=10×10⁻⁹ m, h=1.5×10⁻⁹ m, and n=1.95×10⁻²²moles).

In certain aspects, the percentage of oxygen molecules present in thefluid that are in such nanostructures, or arrays thereof, having acharge-stabilized configuration in the ionic aqueous fluid is apercentage amount selected from the group consisting of greater than:0.1%, 1%; 2%; 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%;65%; 70%; 75%; 80%; 85%; 90%; and greater than 95%. Preferably, thispercentage is greater than about 5%, greater than about 10%, greaterthan about 15% f, or greater than about 20%. In additional aspects, thesubstantial size of the charge-stabilized oxygen-containingnanostructures, or arrays thereof, having a charge-stabilizedconfiguration in the ionic aqueous fluid is a size selected from thegroup consisting of less than: 100 nm; 90 nm; 80 nm; 70 nm; 60 nm; 50nm; 40 nm; 30 nm; 20 nm; 10 nm; 5 nm; 4 nm; 3 nm; 2 nm; and 1 nm.Preferably, this size is less than about 50 nm, less than about 40 nm,less than about 30 nm, less than about 20 nm, or less than about 10 nm.

In certain aspects, the inventive electrokinetic fluids comprisesolvated electrons. In further aspects, the inventive electrokineticfluids comprises charge-stabilized nanostructures and/orcharge-stabilized oxygen-containing nanostructures, and/or arraysthereof, which comprise at least one of: solvated electron(s); andunique charge distributions (polar, symmetric, asymmetric chargedistribution). In certain aspects, the charge-stabilized nanostructuresand/or charge-stabilized oxygen-containing nanostructures, and/or arraysthereof, have paramagnetic properties.

By contrast, relative to the inventive electrokinetic fluids, controlpressure pot oxygenated fluids (non-electrokinetic fluids) and the likedo not comprise such charge-stabilized biologically-activenanostructures and/or biologically-active charge-stabilizedoxygen-containing nanostructures and/or arrays thereof, capable ofmodulation of at least one of cellular membrane potential and cellularmembrane conductivity.

Systems for Making Gas-Enriched Fluids

The presently disclosed system and methods allow gas (e.g. oxygen) to beenriched stably at a high concentration with minimal passive loss. Thissystem and methods can be effectively used to enrich a wide variety ofgases at heightened percentages into a wide variety of fluids. By way ofexample only, deionized water at room temperature that typically haslevels of about 2-3 ppm (parts per million) of dissolved oxygen canachieve levels of dissolved oxygen ranging from at least about 5 ppm, atleast about 10 ppm, at least about 15 ppm, at least about 20 ppm, atleast about 25 ppm, at least about 30 ppm, at least about 35 ppm, atleast about 40 ppm, at least about 45 ppm, at least about 50 ppm, atleast about 55 ppm, at least about 60 ppm, at least about 65 ppm, atleast about 70 ppm, at least about 75 ppm, at least about 80 ppm, atleast about 85 ppm, at least about 90 ppm, at least about 95 ppm, atleast about 100 ppm, or any value greater or therebetween using thedisclosed systems and/or methods. In accordance with a particularexemplary embodiment, oxygen-enriched water may be generated with levelsof about 30-60 ppm of dissolved oxygen.

Table 4 illustrates various partial pressure measurements taken in ahealing wound treated with an oxygen-enriched saline solution (Table 4)and in samples of the gas-enriched oxygen-enriched saline solution ofthe present invention.

TABLE 4 Various partial pressure measurements taken in a healing woundtreated with an inventive saline solution. TISSUE OXYGEN MEASUREMENTSProbe Z082BO In air: 171 mmHg 23° C. Column Partial Pressure (mmHg) B132-36 B2 169-200 B3  20-180* B4 40-60 *wound depth minimal,majority >150, occasional 20 sCosmetic and/or Therapeutic Application and Administration

In particular exemplary embodiments, the gas-enriched fluid of thepresent invention may function as a cosmetic and/or therapeuticcomposition alone or in combination with another cosmetic and/ortherapeutic agent such that the therapeutic composition prevents oralleviates at least one symptom of an eye-related disease or condition.The therapeutic compositions of the present invention includecompositions that are able to be administered to a subject in needthereof. As used herein, “subject,” may refer to any living creature,preferably an animal, more preferably a mammal, and even more preferablya human.

In certain embodiments, the composition formulation may also comprise atleast one additional agent selected from the group consisting of:carriers, adjuvants, emulsifying agents, suspending agents, sweeteners,flavorings, perfumes, and binding agents.

As used herein, “pharmaceutically acceptable carrier” and “carrier”generally refer to a non-toxic, inert solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. Some non-limiting examples of materials which can serve aspharmaceutically acceptable carriers are sugars such as lactose, glucoseand sucrose; starches such as corn starch and potato starch; celluloseand its derivatives such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients such as cocoa butter and suppository waxes; oils suchas peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil;corn oil and soybean oil; glycols; such as propylene glycol; esters suchas ethyl oleate and ethyl laurate; agar; buffering agents such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol, and phosphatebuffer solutions, as well as other non-toxic compatible lubricants suchas sodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator. Inparticular aspects, such carriers and excipients may be gas-enrichedfluids or solutions of the present invention.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, or diluents, are well-known to thosewho are skilled in the art. Typically, the pharmaceutically acceptablecarrier is chemically inert to the therapeutic agents and has nodetrimental side effects or toxicity under the conditions of use. Thepharmaceutically acceptable carriers can include polymers and polymermatrices, nanoparticles, microbubbles, and the like.

In addition to the therapeutic gas-enriched fluid of the presentinvention, the therapeutic composition may further comprise inertdiluents such as additional non-gas-enriched water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. As isappreciated by those of ordinary skill, a novel and improved formulationof a particular therapeutic composition, a novel gas-enrichedtherapeutic fluid, and a novel method of delivering the novelgas-enriched therapeutic fluid may be obtained by replacing one or moreinert diluents with a gas-enriched fluid of identical, similar, ordifferent composition. For example, conventional water may be replacedor supplemented by a gas-enriched fluid produced by infusing oxygen intowater or deionized water to provide gas-enriched fluid.

Certain embodiments provide for therapeutic compositions comprising agas-enriched fluid of the present invention, a pharmaceuticalcomposition or other therapeutic agent or a pharmaceutically acceptablesalt or solvate thereof, and at least one pharmaceutical carrier ordiluent. These pharmaceutical compositions may be used in theprophylaxis and treatment of the foregoing diseases or conditions and intherapies as mentioned above. Preferably, the carrier must bepharmaceutically acceptable and must be compatible with, i.e. not have adeleterious effect upon, the other ingredients in the composition. Thecarrier may be a solid or liquid and is preferably formulated as a unitdose formulation, for example, a tablet which may contain from 0.05 to95% by weight of the active ingredient.

While the compositions and/or methods disclosed herein generally relateto topical application, the most suitable means of administration for aparticular subject will depend on the nature and severity of the diseaseor condition being treated or the nature of the therapy being used, aswell as the nature of the therapeutic composition or additionaltherapeutic agent.

Formulations suitable for topical application include liquids (aqueousor oil based), ointments, creams, lotions, pastes, gels (such ashydrogels), sprays, dispersible powders and granules, emulsions, spraysor aerosols using flowing propellants (such as liposomal sprays, nasaldrops, nasal sprays, and the like) and oils. Suitable carriers for suchformulations include petroleum jelly, lanolin, polyethylene glycol (suchas PEG 3000, PEG 5000, or other), alcohols, and combinations thereof.

Formulations of the invention may be prepared by any suitable method,typically by uniformly and intimately admixing the gas-enriched fluidoptionally with an active compound with liquids or finely divided solidcarriers or both, in the required proportions and then, if necessary,shaping the resulting mixture into the desired shape.

Ointments, pastes, foams, occlusions, creams, gels, sols, suspensions,and patches also can contain excipients, such as starch, tragacanth,cellulose derivatives, silicones, bentonites, silica acid, and talc, ormixtures thereof. Powders and sprays also can contain excipients such aslactose, talc, silica acid, aluminum hydroxide, and calcium silicates,or mixtures of these substances. Solutions of nanocrystallineantimicrobial metals can be converted into aerosols or sprays by any ofthe known means routinely used for making aerosol pharmaceuticals. Ingeneral, such methods comprise pressurizing or providing a means forpressurizing a container of the fluid, usually with an inert carriergas, and passing the pressurized gas through a small orifice. Sprays canadditionally contain customary propellants, such as nitrogen, carbondioxide, or other inert gases. In addition, microspheres ornanoparticles may be employed with the gas-enriched therapeuticcompositions or fluids of the present invention in any of the routesrequired to administer the compounds to a subject.

The topical formulations can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, or gas-enriched fluid, immediately prior touse. Extemporaneous fluids, and/or suspensions can be prepared fromsterile powders, granules, and tablets.

The dose administered to a subject, especially an animal, particularly ahuman, in the context of the present invention should be sufficient toeffect a cosmetic and/or therapeutic response in the animal over areasonable time frame. One skilled in the art will recognize that dosagewill depend upon a variety of factors including a condition of theanimal, the body weight of the animal, the desired outcome, as well asthe condition being treated. A suitable dose is that which will resultin a concentration of the cosmetic and/or therapeutic composition in asubject which is known to affect the desired response. The dosageadministered will, of course, vary depending upon known factors, such asthe pharmacodynamic characteristics of the particular agent and its modeand route of administration; the age, health and weight of therecipient; the nature and extent of the symptoms; the kind of concurrenttreatment; the frequency of treatment; and the effect desired. A dailydosage of active ingredient can be expected to be about 0.0001 to 1000milligrams (mg) per kilogram (kg) of body weight, with the preferreddose being 0.01 to about 30 mg/kg. According to certain aspects dailydosage of active ingredient may be 0.00001 liters to 10 liters, with thepreferred dose being from about 0.0001 liters to 1 liter.

Dosage forms (compositions suitable for administration) contain fromabout 1 mg to about 500 mg of active ingredient per unit. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5-95% weight based on the total weightof the composition.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature, and extentof any adverse side effects that might accompany the administration ofthe therapeutic composition and the desired physiological effect.

The gas-enriched fluids of the present invention may be used to improveexisting drug delivery compositions and methods. The diffuser processedfluids may be formulated, alone or together with one or more therapeuticagents, in suitable dosage unit formulations. In various embodiments,these formulations may include conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants, emulsifying and suspending agents,sweetening, flavoring, perfuming agents, and vehicles appropriate forthe particular route taken into the body of the patient.

In certain embodiments, the gas-enriched fluid disclosed herein maycomprise a wetting, irrigation or soaking fluid to apply to organs, suchas the eye, or into which organs and/or tissues may be placed. One ormore therapeutic agents may be dissolved in the gas-enriched fluid orplaced in the tissue before wetting, irrigating or soaking it in thegas-enriched fluid. The gas-enriched fluid may also be applied to theeye or other organ or tissue in combination with a preexistingmedication, thereby creating a gas-enriched therapeutic fluid, toincrease the efficacy of the medication. The gas-enriched fluid may alsobe used to dissolve a powder, thereby creating a gas-enrichedtherapeutic fluid and drug delivery method. Alternatively, the inventivegas-enriched fluid may comprise infused ingredients of these patches,gels, creams, lotions, ointments, pastes, solutions, sprays, aqueous oroily suspensions, emulsions, and the like.

Topical drug delivery, including drug delivery by administration to theeye or surfaces of the eye is also within the scope of this invention.Particular aspects provide for therapeutic eye care products fordelivery to the eye or to a portion thereof. For example, thegas-enriched diffuser processed fluid may comprise eye ointments, eyedrops, therapeutic solutions, irrigation fluids, pharmaceuticals(prescription and over the counter eye drops and other medications,steroid eye drops, carteol, a non-selective beta blocker, ointment fordry eye, ocular vitamins), and the like. For example, the gas-enrichedfluid may replace common carriers such as saline and water used in eyeointments, eye drops, therapeutic solutions, irrigation fluids and thelike. Alternatively, the inventive gas-enriching diffuser may be used toinfuse one or more of the ingredients of eye ointments, eye drops,therapeutic solution, irrigation fluids and the like into a fluid,thereby producing a novel gas-enriched fluid.

People use contact lenses to correct refractive disorders of the eyesuch as myopia, hyperopia and astigmatism. Lack of oxygen available tothe cornea, due to coverage by the contact lens, results in an increasein microbial infections in contact lens wearers. Thus, certainembodiments herein relate to manufacture of a contact lens utilizing theinventive gas-enriched fluid.

Pseudomonas aeruginosa, and other microbial infections, can causedevastating infections in the human eye and has been associated withneonatal ophthalmia. Pseudomonas can colonize the ocular epithelium bymeans of a fimbrial attachment to sialic acid receptors. If theenvironment is conducive, and particularly if the host's immune systemis compromised, the bacteria can proliferate rapidly via production ofenzymes such as elastase, alkaline protease, and exotoxin A. Infectionwith Pseudomonas can cause loss of use or loss of the entire eye.

An oxygen-enriched fluid may be used to formulate an eye-drop thatpatients can use daily to optimize contact lens wear and thereby provideenhanced levels of oxygen to the oxygen-deprived contact-lens coveredcornea 3508. The gas-enriched fluid produced in accordance with thedisclosed systems and/or methods may be used in the manufacture, storageor care of contact lenses. Heightened levels of dissolved gas mayincrease the potency or safety of fluids, such as solutions used tostore, clean, or moisten contact lenses.

According to various embodiments of the present invention, thegas-enriched inventive fluids disclosed can be used in the manufacture,storage or care of contact lenses (e.g., saline solutions and othercontact lens storage and wetting solutions). The introduction andretention of gases (such as oxygen) to the contact lens may beimplemented during the manufacture and use of contact lenses.Additionally, saline solutions and other contact lens storage andwetting solutions may be produced using gas-enriched fluids describedherein.

Contact lenses are typically formed of soft polymer substances and maygenerally be divided into the categories of hydrophilic and hydrophobiclenses. Hydrophilic contact lenses have a water content in excess of tenpercent while hydrophobic lenses have water content of less than tenpercent. The oxygen permeability of a contact lens depends largely onthe specific polymer used to form the lens. The oxygen permeability maybe increased by using a gas-enriched fluid to hydrate the polymer whenthe lens is created. Contact lenses may be made from a variety ofcommercially available materials, such as hydrophilic polymers (e.g.,hydrogels) or poly(methyl methacrylate). A typical hydrogel polymercomposition may consist of a reaction product of hydrophilicmethacrylamide as well as an acrylic monomer, which may contain azwitterionic monomer, such as a sulfobetaine, for example,N-(3-sulfopropyl)-Nmethacryloxyethyl-N,N-dimethylammonium betaine (SPE),in order to improve the water retention capability.

A variety of oxygen-enriched liquids may be used as eye drops or contactlens solutions. Typical contact lens solutions are made for rinsing,cleaning and disinfecting the contact lenses, including but not limitedto saline solutions and other contact lens storage and wettingsolutions.

The gas-enriched (i.e. oxygenated) solution may be packaged in a bottleor other sealed container provided with a pipette or eye dropper for usein various ocular applications. One such application is that of agas-enriched (i.e. oxygenated) saline solution for use as eye drops orartificial tears. The moisturizing eye drops may be applied directly tothe eye using the eye dropper and applying two or three drops perapplication directly to the eye to alleviate dry eyes, redness, allergicreactions, and to provide additional moisture to the corneal region ofthe eye.

In addition to gas-enriching artificial tears to further supply a gas(e.g. oxygen) to the corneal region, it is also possible to gas-enrichother fluids such as medications that might be applied topically to thesurface of the eye. This may be particularly useful to patients thathave recently undergone surgery on the cornea or other areas of the eyeto improve vision (e.g., laser keratotomy, LASIK, cataract surgery ofall types, extracapsular surgery, phacoemulsification, vitreoretinalsurgery, intraocular lenses and delivery systems, pharmaceuticals,prescription and over the counter eye drops and other medications,steroid eye drops, carteol, a non-selective beta blocker, ointments fordry eye, ocular vitamins and so forth) or to alleviate or lessen theeffects of glaucoma.

It is common to prescribe various antibiotics, anti-inflammatory andpain relieving agents which are applied as drops in solution directly tothe eye itself following these and other surgical procedures. By usinggas-enriched (i.e. oxygenated) aqueous solutions to increase oxygendiffusion into the surface of the eye, it is believed that fasterhealing may occur and that recovery time may be reduced.

Aging eyes often become dry as a result of lowered tear production dueto problems with the tear ducts. This problem is particularly marked inmenopausal women. The most immediate patho-physiological problemproduced by lower tear volume in these patients is the lack of dissolvedoxygen from the air which has only a smaller volume to diffuse to reachthe eye. Using a gas-enriched fluid (such as oxygen-enriched saline) maybe an effective tear supplement. In addition to possible applications inthe form of artificial tears or gas-enriched medicines, thegas-enriching process may also be applied to contact lens solutions suchas saline solutions. A contact lens is normally stored in a solution tokeep the semi-permeable polymer membrane moist and flexible. As shownhere, a contact lens which has been stored in a lens solution isnormally disposed just above the cornea of the eye. The contact lenswill normally float just above the surface of the cornea on a layer ofsolution which may comprise the gas-enriched (i.e. oxygenated) salinesolution in which the lens has been stored. The gas-enriched (i.e.oxygenated) saline solution should increase the amount of gas (e.g.dissolved oxygen) near the cornea of the eye and allow the eye to absorbgreater amounts of oxygen than is usually possible with a contact lensin place.

Surgery:

Particular embodiments provide methods for improved surgery, includingeye surgery procedures, that involve irrigation of tissues (e.g., eyetissues) including but not limited to laser keratomay, LASIK,intraLASIK, cataract surgery, extracapsular surgery,phacoemulsification, intracapsular surgery, vitreoretinal surgery,intraocular lenses and others.

Over 1 million cataract surgeries are performed in the United Stateseach year. Typically, cataract surgeries involve removing the lens, andpossibly receiving an artificial lens. There are three types of surgeryto remove lenses that have a cataract. In extracapsular surgery, thelens is removed while leaving the outer covering of the lens in place;in phacoemulsification, the lens is fragmented by ultrasonic vibrationsand simultaneously irrigated and aspirated, leaving the back half of thelens capsule in place.

Complications of eye surgeries can occur, including high pressure in theeye; blood collection inside the eye; infection inside the eye;artificial lens damage or dislocation; drooping eyelids; retinaldetachment; severe bleeding inside the eye; swelling or clouding of thecornea; blindness; and loss of vision or loss of the entire eye itself.Infection and general tissue health continues to be a challenge in eyesurgeries. Certain embodiments provided herein relate to compositionsand/or methods for improved eye surgery procedures that involveirrigation, and/or moistening, etc., of eye tissues.

According to certain embodiments described herein, compositions and/ormethods are provided for improved surgery, including eye surgeryprocedures that involve irrigation, moistening, etc., of eye tissues.Gas-enriched fluids or solutions of the present invention can be used,for example, for keeping the tissue surface (e.g., ocular surface) moistand/or sterile prior to, during, and/or subsequent to the surgicalprocedure.

Enhancing Biological Tissue Growth:

In other related embodiments, the gas-enriched fluid compositions and/ormethods described herein can be used in relation to enhancing biologicalcell and/or tissue growth of cell (including stem cells), tissue andorgan cultures, whether naturally occurring, or genetically modified. Inaddition, gas-enriched fluid compositions and/or methods disclosedherein can be used for artificial blood and surgical proceduresrequiring artificial blood (such as coronary bypass surgery andshock-trauma procedures). Similarly, oxygen-rich solutions may be usedto perfuse one or more solid organs, such as liver, kidney, heart, eye,hand, foot, brain, and others after harvesting and prior to (e.g. intransit) and during transplantation. Use of gas-enriched (particularlyoxygenated) fluids in accordance with the disclosed embodiments may leadto longer storage time and more successful transplant rates.

In certain embodiments, the inventive fluid may be used for transportingor storing organs, organ samples, test subjects and/or other livingtissues that require or could benefit from the gas-enriched fluidsand/or methods disclosed herein. The gas-enriched (oxygenated) fluid maycontain an organ or other living tissue(s). In certain embodiments, thecontainer may be insulated or provided with a portable refrigerationunit (not shown) and may further include various impellers or othercirculators for moving the oxygen-enriched solution on and about all thesurfaces of the living tissues which are being stored 20 or transportedfor transplantation. In this manner, it is believed that living tissueswill be better preserved with less cell damage prior to and during organor tissue transplantation.

Further embodiments may include systems further comprising a circulationpump which draws fluid and combines the fluid with gas (such as oxygen)from the supply using a diffuser constructed in accordance with thepresent invention. The pump, diffuser, and other components of thetransportation and storage system may be provided with a portable powersupply in the form of one or more batteries or a hydrogen fuel cell.

By storing and transporting organs and other living tissues in agas-enriched storage medium, it is possible to reduce damage to cellsand living tissues outside the body and to supply these tissues totransplantation candidates in a healthier condition. By usinggas-enriched fluids as a storage and transportation medium it ispossible to promote life and health in transplant recipients byintroducing higher levels of dissolved gas(es) (such as oxygen), slowingdown cell decomposition during storage and transportation, and furtherincreasing the probability of a successful organ transplant.

Gas-enriched fluids, such as water, produced in accordance with thedisclosed embodiments may also be used to decontaminate or wash awaycontaminants from a the eye of a subject, such as a person or animal.The gas-enriched fluid compositions and/or methods disclosed hereinprovide high levels of gas(es) (such as oxygen) to the surface beingcleaned (e.g. the eye), which may be particularly therapeutic.

EXAMPLES Example 1 Dissolved Oxygen Stability

As indicated in FIG. 30, there is illustrated the dissolved oxygenlevels in a 500 ml thin-walled plastic bottle and a 1000 ml glass bottlewhich were each capped and stored at 65 degrees Fahrenheit.

As can be seen, when the plastic bottle is opened approximately 65 daysafter bottling, the dissolved oxygen level within the water isapproximately 27.5 ppm. When a second bottle is opened at approximately95 days after bottling, the dissolved oxygen level is approximately 25ppm. Likewise, for the glass bottle, the dissolved oxygen level isapproximately 40 ppm at 65 days and is approximately 41 ppm at 95 days.Thus, this chart indicates that the dissolved oxygen levels within bothplastic and glass bottles are maintained at relatively high rates at 65°Fahrenheit when the oxygen is diffused within the fluid using thedescribed system and method.

Example 2 Decayed Oxygen Content in Balanced Salt Solution

FIG. 33 illustrates the dissolved oxygen retention of a 500 ml balancedsalt solution that originally had a dissolved oxygen level of 5 ppm.Following enrichment of the solution at standard temperature andpressure with the diffuser of the present invention, the dissolvedoxygen level was approximately 41 ppm. The solution was kept in an amberglass bottle. After an hour, the dissolved oxygen level was 40 ppm; 36ppm after two hours; 34 ppm after three hours; and slightly more than 30ppm after approximately four and a half hours. The final measurement wastaken shortly before six hours, at which point the dissolved oxygenlevel was approximately 28 ppm.

Example 3 Microbubble Size

Experiments were performed with a gas-enriched fluid by using thediffuser of the present invention in order to determine a gasmicrobubble size limit. The microbubble size limit was established bypassing the gas enriched fluid through 0.22 and 0.1 micron filters. Inperforming these tests, a volume of fluid passed through the diffuser ofthe present invention and generated a gas-enriched fluid. Sixtymilliliters of this fluid was drained into a 60 ml syringe. Thedissolved oxygen level of the fluid within the syringe was then measuredby Winkler titration. The fluid within the syringe was injected througha 0.22 micron Millipore Millex GP50 filter and into a 50 ml beaker. Thedissolved oxygen rate of the material in the 50 ml beaker was thenmeasured. The experiment was performed three times to achieve theresults illustrated in Table 5 below.

TABLE 5 DO AFTER 0.22 MICRON DO IN SYRINGE FILTER 42.1 ppm 39.7 ppm 43.4ppm 42.0 ppm 43.5 ppm 39.5 ppm

As can be seen, the dissolved oxygen levels that were measured withinthe syringe and the dissolved oxygen levels within the 50 ml beaker werenot significantly changed by passing the diffused material through a0.22 micron filter, which implies that the microbubbles of dissolved gaswithin the fluid are not larger than 0.22 microns.

A second test was performed in which a batch of saline solution wasenriched with the diffuser of the present invention and a sample of theoutput solution was collected in an unfiltered state. The dissolvedoxygen level of the unfiltered sample was 44.7 ppm. A 0.1 micron filterwas used to filter the oxygen-enriched solution from the diffuser of thepresent invention and two additional samples were taken. For the firstsample, the dissolved oxygen level was 43.4 ppm. For the second sample,the dissolved oxygen level was 41.4 ppm. Finally, the filter was removedand a final sample was taken from the unfiltered solution. In this case,the final sample had a dissolved oxygen level of 45.4 ppm. These resultswere consistent with those in which the Millipore 0.22 micron filter wasused. Thus, the majority of the gas bubbles or microbubbles within thesaline solution are approximately less than 0.1 microns in size.

Example 4 Sparging Effects

FIGS. 34 and 35 illustrate the sparging affects of the diffuser of thepresent invention on a fluid passing therethrough. The sparging ofoxygen-enriched water occurred in an 8 gallon tank at standardtemperature and pressure. As indicated, initially the oxygen-enrichedwater had a dissolved oxygen level of approximately 42 ppm. After 2minutes of running through the diffuser, the nitrogen had sparged theoxygen-enriched water such that the dissolved oxygen level was thenslightly more than 20 ppm. At 6 minutes, the dissolved oxygen level wasapproximately 6 ppm. The dissolved oxygen level of the oxygen-enrichedwater reached a minimum value slightly greater than zero (0) atapproximately 14 minutes after the beginning of the process. Thesefigures illustrate the manner in which nitrogen may be diffused intowater to sparge the oxygen from the water. However, any gas could beused within any fluid to sparge one gas from the other and diffuse theother gas into the fluid. The same experiment could utilize any hostfluid material, and any fluid infusion material.

Example 5 Rayleigh Effects

Fluids processed through the diffuser device described herein exhibitdifferences within the structure of the water when compared with normalunprocessed water. Gas-enriched water made by embodiments disclosedherein has been shown to have more Rayleigh scattering compared tounprocessed water.

In experiments conducted, samples of gas-enriched and non-enriched waterwere prepared and sent for optical analysis. The purpose of these testswas to determine whether there are any gross optical differences betweennormal (unprocessed) deionized water and water enriched by the diffuserdevice of the present invention.

The two samples, were coded to maintain their identities in secrecy, andonly after the tests were completed were the samples identified. The twosamples were placed in a laser beam of 633 nanometers according to thediagram illustrated in FIG. 37A. Sample B, which was gas-enriched fluidaccording to certain embodiments disclosed herein, exhibited scatteredlight regardless of its position relative to the laser source. TheSample B fluid had been sealed in glass bottles for approximately oneweek. After two to three hours of opening the bottle, the scatteringeffect disappeared. Thus, the structure of the gas-enriched fluid isoptically different from the structure of the unprocessed fluid. Theoptical effect is not directly related to dissolved oxygen levels sincethe dissolved oxygen level at the start was approximately 45 ppm and atthe end of the experiment was estimated to be approximately 32 ppm.Results are shown in FIG. 37B.

Example 6 Generation of Solvated Electrons

Additional evidence has also suggested that the enriching processgenerated by the diffuser device of the present invention results insolvated electrons within the gas-enriched fluid. Due to the results ofthe polarographic dissolved oxygen probes, it is believed that thediffused fluid exhibits an electron capture effect and thus the fluidmay include solvated electrons within the gas-enriched material.

There are two fundamental techniques for measuring dissolved oxygenlevels electrically: galvanic measuring techniques and polarographicmeasurements. Each process uses an electrode system wherein thedissolved oxygen levels within the solution being tested react with acathode of the probe to produce a current. Dissolved oxygen levelsensors consist of two electrodes, an anode and a cathode, which areboth immersed in electrolyte within the sensor body. An oxygen permeablemembrane separates the anode and cathode from the solution being tested.Oxygen diffuses across the membrane and interacts with the internalcomponents of the probe to produce an electrical current. The cathode isa hydrogen electrode and carries negative potential with respect to theanode. The electrolyte solution surrounds the electrode pair and iscontained by the membrane. When no oxygen is present, the cathode ispolarized by hydrogen and resists the flow of current. When oxygenpasses through the membrane, the cathode is depolarized and electronsare consumed. The cathode electrochemically reduces the oxygen tohydroxyl ions according to the following equation:O₂+2H₂O+4E⁻=4OH⁻

When performing dissolved oxygen level measurements of a gas-enrichedsolution according to the systems of the present invention, an overflowcondition has been repeatedly experienced wherein the dissolved oxygenmeter displays a reading that is higher than the meter is capable ofreading. However, evaluation of the gas-enriched solution by WinklerTitration indicates lower dissolved oxygen (DO) level for the solutionthan indicated by the probe. Typically, a DO probe (such as the Orion862 used in these experiments) has a maximum reading of 60 ppm. However,when the meter is left in gas-enriched water of the present invention,it overflows.

Without wishing to be bound by any particular mechanism of action, themechanism of the meter responds to electrons where the oxygen reacts.However, according to electron spin resonance, no free ions are presentin the fluid. Thus, the fluid presumably contains solvated electronsstabilized by the oxygen species that is also present in the fluid.

Example 7 In Vitro Wound Healing

The effects of a gas-enriched fluid (enriched with oxygen) were testedfor the ability of cultured human epidermal keratinocytes to seal awound.

Human epidermal keratinocytes were isolated from neonatal foreskins thatwere obtained from routine circumcision and de-identified. Foreskinswere washed twice in PBS and incubated in 2.4 U/mL Dispase II in orderto separate the dermis from the epidermis. The epidermis was incubatedwith 0.25% trypsin/1 mM EDTA, neutralized with soy bean trypsininhibitor, agitated, and passed through a 70 um sieve to separate thecells. Next, the cell suspension was centrifuged and resuspended in cellculture medium (M154) supplemented with 0.07 mM CaCl₂, and humankeratinocyte growth supplements (0.2% hydrocortisone, 0.2 ng/mL humanepidermal growth factor) and penicillin/streptomycin, amphoteracinantibiotic cocktail. The keratinocyte cell suspensions were plated ontouncoated 12-well culture dishes and the medium replaced after 24 hours,and every 48 hours after the initial seeding.

Upon reaching cellular confluence, linear scratches were made with asterile p1000 pipette tip, which resulted in a uniform cell-free wound.The monolayers were washed several times with Dulbecco's PBS in order toremove any cellular debris. The wound monolayers were then incubated inthe following media: i) the complete growth media (as described above inthis Example); ii) the complete growth media diluted 1:1 with a shearedversion of saline without oxygen (control fluid that was processed usingthe disclosed diffuser device but without adding a gas); and iii) thecomplete growth media diluted 1:1 with oxygen-enriched saline. Eachstudy was done in triplicate.

Prior to incubation, the wells were filled with the respective media andsealed by placing a 25×25 mm glass coverslip on top of each well. At 6,12, 24, and 48 hours post-wounding, oxygen measurements were made, andcultures were imagined.

Six hours post-wounding, the edges of the wounds in the saline andgas-enriched media were more ruffled than those in the media controlthat was processed with the diffuser device disclosed herein, butwithout the addition of a gas. Twelve hours post-wounding the edges ofthe wounds in all three media appeared uneven, with keratinocytes alongthe borders migrating toward the center of the wounds. Quantification ofmigrating keratinocytes revealed approximately the same level ofkeratinocyte migration in the saline and gas-enriched media. Results ofthe experiment are shown in FIGS. 40A and 44B.

Example 8 Improved Wound Healing

A study was performed to determine the improved healing characteristicsof wounds that were exposed to an oxygen-enriched saline solution thatwas processed according to embodiments disclosed herein. In thisexperiment, bandages were placed on porcine dermal excision biopsywounds. The bandages soaked in oxygen-enriched saline solution or acontrol group of bandages soaked in a saline solution that was notoxygen-enriched. Microscopically, several factors were evaluated by thestudy including: 1) epidermalization; 2) neovascularization; 3)epidermal differentiation; 4) mast cell migration; and 5) mitosis.

Externally, the wounds appeared to heal at varying rates. The woundstreated with the oxygen-enriched saline solution showed an increase inwound healing at days 4 through 11. However, both wounds seemed tocomplete healing at approximately the same time. The study showed thatbetween days 3 and 11, the new epidermis in wounds treated with theoxygen-enriched saline solution migrated at two to four times as fast asthe epidermis of the wounds treated with the normal saline solution. Thestudy also showed that between 15 and 22 days, the wound treated by theoxygen-enriched saline solution differentiated at a more rapid rate asevidenced by the earlier formation of more mature epidermal layers. Atall stages, the thickening that occurs in the epidermis associated withnormal healing did not occur within the wounds treated by theoxygen-enriched saline solution.

Without wishing to be bound by any particular theory, it is believedthat the oxygen-enriched saline solution may increase the localizedlevel of NO within the wounds. NO modulates growth factors, collagendeposition, inflammation, mast cell migration, epidermal thickening, andneovascularization in wound healing. Furthermore, nitric oxide isproduced by an inducible enzyme that is regulated by oxygen.

Thus, while not wishing to be bound to any particular theory, theinventive gas-enriched fluid may stimulate NO production, which is inaccordance with the spectrum of wound healing effects seen in theseexperiments.

The epidermis of the healing pigs experienced earlier differentiation inthe oxygen-enriched saline group at days 15 through 22. In the case ofmast cell migration, differences also occurred in early and latemigration for the oxygen-enriched solution. A conclusive result for thelevel of mitosis was unascertainable due to the difficulty in staining.

Referring now to FIG. 41A through 41F, various illustrations compare thewound healing results of the porcine epidermal tissues with or withoutoxygen-enriched saline solution. Thus, the healing of the control woundand of the wound using the oxygen-enriched saline solution was followedfor days 1, 4 and 16. FIG. 41A illustrates the wound healing for thecontrol wound on day 1. As can be seen, the wound shows epidermal/dermalthickening and a loss of contour. FIG. 41B illustrates the wound healingon day 1 for the wound treated using the oxygen-enriched salinesolution. The wound shows normal epidermal/dermal thickness and normalcontouring is typical on a new wound.

Referring now to FIGS. 41C and 41D, there are illustrated the woundhealing for the control wound on day 4 and the wound healing for thewound treated with the oxygen-enriched saline solution on day 4. For thecontrol wound illustrated in FIG. 41C, the wound shows a 600 micronepidermal spur. In the wound treated with the oxygen-enriched salinesolution in FIG. 41D, there is illustrated a 1200 micron epidermal spur.Thus, in the first 4 days of the experiment, the epidermal spur createdin the wound treated using the oxygen-enriched saline solution shows anepidermal growth rate of twice of that of the wound that was not treatedwith the oxygen-enriched saline solution.

Referring now to FIG. 41E, there is illustrated the control wound at day16. The wound shows less differentiated epidermis with loss ofepidermal/dermal contour than that illustrated by the wound treated withthe oxygen-enriched saline solution illustrated in FIG. 41F. FIG. 41Fshows more differentiated epidermis and more normal epidermal/dermalcontouring in the wound.

Thus, as illustrated with respect to FIGS. 41A through 41F, the woundtreated with the oxygen-enriched saline solution shows much greaterhealing characteristics than the untreated wound and shows a greaterdifferentiated epidermis with more normal epidermal/dermal contour.

Example 9 Glutathione Peroxidase Study

The inventive oxygen-enriched fluid was tested for the presence ofhydrogen peroxide by testing the reactivity with glutathione peroxidaseusing a standard assay (Sigma). Water samples were tested by adding theenzyme cocktail and inverting. Continuous spectrophotometric ratedetermination was made at A₃₄₀ nm, and room temperature (25 degreesCelsius). Samples tested were: 1. deionized water (negative control), 2.inventive oxygen-enriched fluid at low concentration, 3. inventiveoxygen-enriched fluid at high concentration, 4. hydrogen peroxide(positive control). The hydrogen peroxide positive control showed astrong reactivity, while none of the other fluids tested reacted withthe glutathione peroxidase.

Example 10 Electrokinetically Generated Superoxygenated Fluids and SolasWere Shown to Provide for Synergistic Prolongation Effects (e.g.,Suppression of Bronchoconstriction) with Albuterol in vivo in anArt-Recognized Animal Model of Human Bronchoconstriction (Human AsthmaModel)

Experiment 1:

In an initial experiment, sixteen guinea pigs were evaluated for theeffects of bronchodilators on airway function in conjunction withmethacholine-induced bronchoconstriction. Following determination ofoptimal dosing, each animal was dosed with 50 μg/mL to deliver thetarget dose of 12.5 μg of albuterol sulfate in 250 μL per animal.

The study was a randomized blocked design for weight and baseline PenHvalues. Two groups (A and B) received an intratracheal instillation of250 μL of 50 μg/mL albuterol sulfate in one or two diluents: Group A wasdeionized water that had passed through the inventive device, withoutthe addition of oxygen, while Group B was inventive gas-enriched water.Each group was dosed intratracheally with solutions using a Penn CenturyMicrosprayer. In addition, the animals were stratified across BUXCOplethysmograph units so that each treatment group is represented equallywithin nebulizers feeding the plethysmographs and the recording units.

Animals that displayed at least 75% of their baseline PenH value at 2hours following albuterol administration were not included in the dataanalyses. This exclusion criteria is based on past studies where thefailure to observe bronchoprotection with bronchodilators can beassociated with dosing errors. As a result, one animal from the controlgroup was dismissed from the data analyses.

Once an animal had greater than 50% bronchoconstriction, the animal wasconsidered to be not protected. As set forth in Table 6 below, 50% ofthe Group B animals (shaded) were protected from bronchoconstriction outto 10 hours (at which time the test was terminated).

TABLE 6 Bronchoconstriction Protection as Measured with MethacholineChallenge

Experiment 2: A Bronchoconstriction Evaluation of RDC1676 with AlbuterolSulfate in Male Hartley Guinea Pigs:

An additional set of experiments was conducted using a larger number ofanimals to evaluate the protective effects of the inventiveelectrokinetically generated fluids (e.g, RDC1676-00, RDC1676-01,RDC1676-02 and RDC1676-03) against methacholine-inducedbronchoconstriction when administered alone or as diluents for albuterolsulfate in male guinea pigs.

Materials:

Guinea Pigs (Cavia porcellus) were Hartley albino, Crl:(HA)BR fromCharles River Canada Inc. (St. Constant, Quebec, Canada). Weight:Approximately 325±50 g at the onset of treatment. Number of groups was32, with 7 male animals per group (plus 24 spares form same batch ofanimals). Diet; All animals had free access to a standard certifiedpelleted commercial laboratory diet (PMI Certified Guinea Pig 5026; PMINutrition International Inc.) except during designated procedures.

Methods:

Route of administration was intratracheal instillation via a PennCentury Microsprayer and methacholine challenge via whole bodyinhalation. The intratracheal route was selected to maximize lungexposure to the test article/control solution. Whole body inhalationchallenge has been selected for methacholine challenge in order toprovoke an upper airway hypersensitivity response (i.e.bronchoconstriction).

Duration of treatment was one day.

Table 7 shows the experimental design. All animals were subjected toinhalation exposure of methacholine (500 μg/ml), 2 hours followingTA/Control administration. All animals received a dose volume of 250 μl.Therefore, albuterol sulfate was diluted (in the control article and the4 test articles) to concentrations of 0, 25, 50 and 100 μg/ml.

Thirty minutes prior to dosing, solutions of albuterol sulfate of 4different concentrations (0, 25, 50 and 100 μg/ml) was made up in a l Oxstock (500 μg/mL) in each of these four test article solutions(RDC1676-00, RDC1676-01, RDC1676-02; and RDC1676-03). Theseconcentrations of albuterol sulfate were also made up innon-electrokinetically generated control fluid (control 1). The dosingsolutions were prepared by making the appropriate dilution of each stocksolution. All stock and dosing solutions were maintained on ice onceprepared. The dosing was completed within one hour after thetest/control articles are made. A solution of methacholine (500 μg/ml)was prepared on the day of dosing.

Each animal received an intratracheal instillation of test or controlarticle using a Penn Century microsprayer. Animals were food deprivedovernight and were anesthetized using isoflurane, the larynx wasvisualized with the aid of a laryngoscope (or suitable alternative) andthe tip of the microsprayer was inserted into the trachea. A dose volumeof 250 μl/animal of test article or control was administered.

The methacholine aerosol was generated into the air inlet of a mixingchamber using aeroneb ultrasonic nebulizers supplied with air from aBuxco bias flow pump. This mixing chamber in turn fed four individualwhole body unrestrained plethysmographs, each operated under a slightnegative pressure maintained by means of a gate valve located in theexhaust line. A vacuum pump was used to exhaust the inhalation chamberat the required flow rate.

Prior to the commencement of the main phase of the study, 12 spareanimals were assigned to 3 groups (n=4/group) to determine the maximumexposure period at which animals may be exposed to methacholine toinduce a severe but non-fatal acute bronchoconstriction. Four animalswere exposed to methacholine (500 μg/mL) for 30 seconds and respiratoryparameters were measured for up to 10 minutes following commencement ofaerosol. Methacholine nebulizer concentration and/or exposure time ofaerosolization was adjusted appropriately to induce a severe butnon-fatal acute/reversible bronchoconstriction, as characterized by antransient increase in penes.

Once prior to test article administration (Day −1) and again at 2, 6,10, 14, 18, 22 and 26 hours postdose, animals were placed in the chamberand ventilatory parameters (tidal volume, respiratory rate, derivedminute volume) and the enhanced pause Penh were measured for a period of10 minutes using the Buxco Electronics BioSystem XA system, followingcommencement of aerosol challenge to methacholine. Once animals werewithin chambers baseline, values were recorded for 1-minute, followingwhich methacholine, nebulizer concentration of 500 ug/mL wereaerosoloized for 30 seconds, animals were exposed to the aerosol forfurther 10 minutes during which time ventilatory paramaters werecontinuously assessed. Penh was used as the indicator ofbronchoconstriction; Penh is a derived value obtained from peakinspiratory flow, peak expiratory flow and time of expiration.Penh=(Peak expiratory flow/Peak inspiratory flow)*(Expiratory time/timeto expire 65% of expiratory volume−1).

Animals that did not display a severe acute broncoconstriction duringthe predose methacholine challenge were replaced. Any animal displayingat least 75% of their baseline PenhPenes value at 2 hours post dose werenot included in the data analysis. The respiratory parameters wererecorded as 20 second means.

Data considered unphysiological was excluded from further analysis.

Changes in Penh were plotted over a 15 minute period and Penh value wasexpressed as area under the curve. Numerical data was subjected tocalculation of group mean values and standard deviations (asapplicable).

TABLE 7 Experimental design; 7 male guinea pigs per group. AlbuterolAlbuterol Albuterol Albuterol (0 μg/ (6/25 μg/ (12.5 μg/ (25 μg/ GroupID animal) animal) animal) animal) 1 (control 1) 7 males 7 males 7 males7 males (ambient oxygen) 5 (RDC1676-00 7 males 7 males 7 males 7 males(Solas) 6 (RDC1676-01 7 males 7 males 7 males 7 males (20 ppm oxygen) 7(RDC1676-02 7 males 7 males 7 males 7 males (40 ppm oxygen) 8(RDC1676-03 7 males 7 males 7 males 7 males (60 ppm oxygen)Results:

As shown in FIG. 107A-D, in the absence of Albuterol, administration ofthe inventive electrokinetically generated fluids had no apparent effecton mean percent baseline PenH values, when measured over a 26 hourperiod.

Surprisingly, however, as shown in FIG. 108A-D, administration ofalbuterol (representative data for the 25 μg albuterol/animal groups areshown) formulated in the inventive electrokinetically generated fluids(at all oxygen level values tested; ambient (FIG. 108-A), 20 ppm (FIG.108-B), 40 ppm (FIG. 108-C) and 60 ppm (FIG. 108-D)) resulted in astriking prolongation of anti-broncoconstrictive effects of albuterol,compared to control fluid. That is, the methacholine results showed aprolongation of the bronchodilation of albuterol out to at least 26hours. FIGS. 108A-D shows that there were consistent differences at alloxygen levels between RDC1676 and the normal saline control. Combiningall 4 RDC1676 fluids, the p value for the overall treatment differencefrom normal saline was 0.03.

According to particular aspects of the present invention, therefore, theinventive electrokinetically generated solutions provide for synergisticprolongation effects with Albuterol, thus providing for a decrease in apatient's albuterol usage, enabling more efficient cost-effective druguse, fewer side effects, and increasing the period over which a patientmay be treated and responsive to treatment with albuterol.

Example 11 A Cytokine Profile was Determined

Mixed lymphocytes were obtained from a single healthy volunteer donor.Buffy coat samples were washed according to standard procedures toremove platelets. Lymphocytes were plated at a concentration of 2×10⁶per plate in RPMI media (+50 mm HEPES) diluted with either inventivegas-enriched fluid or distilled water (control). Cells were stimulatedwith 1 microgram/mL T3 antigen, or 1 microgram/mL phytohemagglutinin(PHA) lectin (pan-T cell activator), or unstimulated (negative control).Following 24-hour incubation, cells were checked for viability and thesupernatants were extracted and frozen.

The supernatants were thawed, centrifuged, and tested for cytokineexpression using a XMAP® (Luminex) bead lite protocol and platform.

Two million cells were plated into 6 wells of a 24-well plate in fullRPMI+50 mm Hepes with either inventive oxygen-enriched fluid (water)(wells 1, 3, and 5) or distilled water (2, 4 and 6) (10×RPMI dilutedinto water to make 1×). Cells were stimulated with 1 ug/ml T3 antigen(wells 1 and 2) or PHA (wells 3 and 4). Control wells 5 and 6 were notstimulated. After 24 hours, cells were checked for viability andsupernatants were collected and frozen. Next, the supernatants werethawed and spun at 8,000 g to pellet. The clarified supernatants wereassayed for the cytokines listed using a LUMINEX BEAD LITE™ protocol andplatform. The numerical data is tabulated in Table 8, and thecorresponding bar graphs are depicted in FIG. 38. Notably, IFN-γ levelwas higher in the inventive gas-enriched culture media with T3 antigenthan in the control culture media with T3 antigen, while IL-8 was lowerin the inventive gas-enriched culture media with T3 antigen than in thecontrol culture media with T3 antigen. Additionally, IL-6, IL-8, andTNF-α levels were lower in the inventive gas-enriched media with PHA,than in the control media with PHA, while IL-1β levels were lower in theinventive gas-enriched fluid with PHA when compared with control mediawith PHA. In the inventive gas-enriched media alone. IFN-γ levels werehigher than in control media.

TABLE 8 Sample IFN II-10 II-12p40 II-12p70 II-2 II-4 II-5 II-6 II-8II-ib IP-10 TNFa 1 0 0 0 2.85 0 0 7.98 20.3 1350 7.56 11500 15.5 2 0 0 03.08 0 0 8 15.2 8940 3.68 4280 7.94 3 0 581 168 3.15 0 0 8 16400 22003280 862 13700 4 0 377 56.3 4.22 0 0 8.08 23800 22100 33600 558 16200 50 0 0 2.51 0 0 7.99 24 1330 7.33 5900 8.55 6 0 0 0 2.77 0 0 8 5.98 32104.68 3330 0

Example 12 Myelin Oligodendrocyte Glycoprotein (MOG)

As set forth in FIG. 48, lymphocyte proliferation in response to MOGantigenic peptide was increased when cultured in the presence of theinventive gas-enriched fluid when compared to pressurized, oxygenatedfluid (pressure pot) or deionized control fluid. Thus, the inventivegas-enriched fluid amplifies the lymphocyte proliferative response to anantigen to which the cells were previously primed. Myelinoligodendrocyte glycoprotein peptide 35-55 (MOG 35-55)(M-E-V-G-W-Y-R-S-P-F-S-R-O-V-H-L-Y-R-N-G-K) (SEQ ID NO:1; seepublication US20080139674, incorporated by reference herein, includingfor purposes of this SEQ ID NO:1) corresponding to the known mousesequence was synthesized. Next, 5×10⁵ spleen cells were removed from MOGT cell receptor transgenic mice previously immunized with MOG, and werecultured in 0.2 ml TCM fluid reconstituted with inventive gas-enrichedfluid, pressurized oxygenated water (pressure pot water) or with controldeionized water. Splenocytes were cultured with MOG p35-55 for 48 or 72hours, respectively. Cultures were pulsed with 1 Ci [3H]-thymidine andharvested 16 hours later. Mean cpm of [3H] thymidine incorporation wascalculated for triplicate cultures. Results are shown in FIG. 48.

Example 13 Cytokine Expression

In particular aspects, human mixed lymphocytes were stimulated with T3antigen or PHA in inventive electrokinetic fluid, or control fluid, andchanges in IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40),IL-12(p70), IL-13, IL-17, Eotaxin, IFN-γ, GM-CSF, MIP-1β, MCP-1, G-CSF,FGFb, VEGF, TNF-α, RANTES, Leptin, TNF-β, TFG-β, and NGF were evaluated.As can be seen from FIG. 38, pro-inflammatory cytokines (IL-1β, TNF-α,IL-6, and GM-CSF), chemokines (IL-8, MIP-1α, RANTES, and Eotaxin),inflammatory enzymes (iNOS, COX-2, and MMP-9), allergen responses (MHCclass II, CD23, B7-1, and B7-2), and Th2 cytokines (IL-4, IL-13, andIL-5) tested were reduced in test fluid versus control fluid. Bycontrast, anti-inflammatory cytokines (e.g., IL1R-α, TIMPs) tested wereincreased in test fluid versus control fluid.

To expand on these data, Applicants used an art recognized model systeminvolving ovalbumin sensitization, for assessing allergichypersensitivity reactions. The end points studied were particularcytologic and cellular components of the reaction as well as serologicmeasurements of protein and LDH. Cytokine analysis was performed,including analysis of Eotaxin, IL-1A, IL-1B, KC, MCP-1, MCP-3, MIP-1A,RANTES, TNF-A, and VCAM.

Briefly, male Brown Norway rats were injected intraperitoneally with 0.5mL Ovalbumin (OVA) Grade V (A5503-1G, Sigma) in solution (2.0 mg/mL)containing aluminum hydroxide (Al(OH)₃) (200 mg/mL) once each on days 1,2, and 3. The study was a randomized 2×2 factorial arrangement oftreatments (4 groups). After a two week waiting period to allow for animmune reaction to occur, the rats were either exposed or were treatedfor a week with either RDC1676-00 (sterile saline processed through theRevalesio proprietary device), and RDC1676-01 (sterile saline processedthrough the Revalesio proprietary device with additional oxygen added).At the end of the 1 week of treatment for once a day, the 2 groups werebroken in half and 50% of the rats in each group received either Salineor OVA challenge by inhalation.

Specifically, fourteen days following the initial serialization, 12 ratswere exposed to RDC 1676-00 by inhalation for 30 minutes each day for 7consecutive days. The air flow rate through the system was set at 10liters/minute. A total of 12 rats were aligned in the pie chamber, witha single port for nebulized material to enter and evenly distribute tothe 12 sub-chambers of the Aeroneb.

Fifteen days following initial sensitization, 12 rats were exposed toRDC 1676-01 by ultrasonic nebulization for 30 minutes each day for 7consecutive days. The air flow was also set for 10 liters/minute, usingthe same nebulizer and chamber. The RDC 1676-00 was nebulized first andthe Aeroneb chamber thoroughly dried before RDC 1676-01 was nebulized.

Approximately 2 hours after the last nebulization treatment, 6 rats fromthe RDC 1676-00 group were re-challenged with OVA (1% in saline)delivered by intratreacheal instillation using a Penn CenturyMicrosprayer (Model 1A-1B). The other 6 rats from the RDC 1676-00 groupwere challenged with saline as the control group delivered by way ofintratreacheal instillation. The following day, the procedure wasrepeated with the RDC 1676-01 group.

Twenty four hours after re-challenge, all rats in each group wereeuthanized by overdose with sodium pentobarbital. Whole blood sampleswere collected from the inferior vena-cava and placed into two disparateblood collection tubes: Qiagen PAXgene™ Blood RNA Tube and QiagenPAXgene™ Blood DNA Tube. Lung organs were processed to obtainbronchoalveolar lavage (BAL) fluid and lung tissue for RT-PCR to assesschanges in markers of cytokine expression known to be associated withlung inflammation in this model. A unilateral lavage technique was beemployed in order to preserve the integrity of the 4 lobes on the rightside of the lung. The left “large” lobe was lavaged, while the 4 rightlobes were tied off and immediately placedinot TRI-zol™, homogenized,and sent to the lab for further processing.

BAL Analysis.

Lung lavage was collected and centrifuged for 10 minutes at 4° C. at600-800 g to pellet the cells. The supernatants were transferred tofresh tubes and frozen at −80° C. Bronchial lavage fluid (“BAL”) wasseparated into two aliquots. The first aliquot was spun down, and thesupernatant was snap frozen on crushed dry ice, placed in −80° C., andshipped to the laboratory for further processing. The amount of proteinand LDH present indicates the level of blood serum protein (the proteinis a serum component that leaks through the membranes when it'schallenged as in this experiment) and cell death, respectively. Theproprietary test side showed slight less protein than the control.

The second aliquot of bronchial lavage fluid was evaluated for totalprotein and LDH content, as well as subjected to cytologicalexamination. The treated group showed total cells to be greater than thesaline control group. Further, there was an increase in eosinophils inthe treated group versus the control group. There were also slightlydifferent polymorphonuclear cells for the treated versus the controlside.

Blood Analysis.

Whole blood was analyzed by transfer of 1.2-2.0 mL blood into a tube,and allowing it to clot for at least 30 minutes. The remaining bloodsample (approximately 3.5-5.0 mL) was saved for RNA extraction usingTRI-zol™ or PAXgene™. Next, the clotted blood sample was centrifuged for10 minutes at 1200 g at room temperature. The serum (supernatant) wasremoved and placed into two fresh tubes, and the serum was stored at−80° C.

For RNA extraction utilizing Tri-Reagent (TB-126, Molecular ResearchCenter, Inc.), 0.2 mL of whole blood or plasma was added to 0.75 mL ofTRI Reagent BD supplemented with 20 μL of 5N acetic acid per 0.2 mL ofwhole blood or plasma. Tubes were shaken and stored at −80° C. UtilizingPAXgene™, tubes were incubated for approximately two hours at roomtemperature. Tubes were then placed on their side and stored in the −20°C. freezer for 24 hours, and then transferred to −80° C. for long termstorage.

Luminex Analysis.

By Luminex platform, a microbead analysis was utilized as a substratefor an antibody-related binding reaction which is read out in luminosityunits and can be compared with quantified standards. Each blood samplewas run as 2 samples concurrently. The units of measurement areluminosity units and the groups are divided up into OVA challengedcontrols, OVA challenged treatment, and saline challenged treatment withproprietary fluid.

For Agilant gene array data generation, lung tissue was isolated andsubmerged in TRI Reagent (TR118, Molecular Research Center, Inc.).Briefly, approximately 1 mL of TRI Reagent was added to 50-100 mg oftissue in each tube. The samples were homogenized in TRI Reagent, usingglass-Teflon™ or Polytron™ homogenizer. Samples were stored at −80° C.

Blood Samples:

FIGS. 49-58 show the results of whole blood sample evaluations.

Exemplary FIG. 49 shows the basic luminosity data presentation formatfor the blood sample data. Letters designating the identity of themeasured cytokine (in this case KC) are at the top right of each datafigure. The data is presented both as data points (upper graph) and bargraphs (lower graph) of the individual samples. In either case, thegraphs are divided, from left to right, in four groups. The first 2groups (RDC1676-00 OVA and RDC1676-01 OVA, respectively) were those thatwere re-challenged with OVA by inhalation, whereas the last two groups(RDC1676-00 OVA and RDC1676-01 OVA, respectively) where those that werere-challenged with saline control only. Again, the suffix 00 representssaline treatment and suffix 01 represents inventive electrokinetic fluidtreated groups.

Each blood sample was split into 2 samples and the samples were runconcurrently. The units of measure are units of luminosity and thegroups, going from left to right are: OVA challenged controls; OVAchallenged inventive electrokinetic fluid treatment; followed by salinechallenged saline treatment; and saline challenged inventiveelectrokinetic fluid treatment. To facilitate review, both theRDC1676-01 groups are highlighted with gray shaded backdrops, whereasthe control saline treatment groups have unshaded backdrops.

Generally, in comparing the two left groups, while the spread of theRDC1676-01 group data is somewhat greater, particular cytokine levels inthe RDC1676-01 group as a whole are less than the samples in the controltreated group; typically about a 30% numerical difference between the 2groups. Generally, in comparing the right-most two groups, theRDC1676-01 group has a slightly higher numerical number compared to theRDC1676-00 group.

FIG. 50 shows analysis of RANTES (IL-8 super family) in blood sampledata according to particular exemplary aspects. Luminosity units for theleftmost two groups (the OVA challenged groups) indicate that generallyvalues in the RDC1676-01 treated group were less than the RDC1676-00control group as shown by the dot plot in the upper graph portion whichagain shows a 30-35% differential between the two groups, whereas in thesaline only exposed groups the cytokine level values where roughly thesame, or perhaps slightly increased in the RDC1676-01 treated group.

FIG. 51 shows analysis of MCP-1 in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 52 shows analysis of TNF alpha in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 53 shows analysis of MIP-1 alpha in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 54 shows analysis of IL-1 alpha in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 55 shows analysis of Vcam in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIG. 56 shows analysis of IL-1 beta in blood sample data according toparticular exemplary aspects. Luminosity units for the leftmost twogroups (the OVA challenged groups) indicate that generally values in theRDC1676-01 treated group were less than the RDC1676-00 control group asshown by the dot plot in the upper graph portion, whereas in the salineonly exposed groups the cytokine level values where roughly the same, orperhaps slightly increased in the RDC1676-01 treated group.

FIGS. 57 and 58 show analysis of Eotaxin and MCP-3, respectively, inblood sample data according to particular exemplary aspects. In eachcase, luminosity units for the leftmost two groups (the OVA challengedgroups) indicate that generally values in the RDC1676-01 treated groupwere less than the RDC1676-00 control group as shown by the dot plot inthe upper graph portion, whereas in the saline only exposed groups thecytokine level values where roughly the same, or perhaps slightlyincreased in the RDC1676-01 treated group.

Bronchial Lavage Samples:

FIGS. 59-68 show the corresponding results of bronchoalveolar lavagefluid (BAL) sample evaluations.

FIG. 59 shows analysis of KC in BAL data according to particularexemplary aspects. In this instance the response level, coupled withsampling variability, was inconclusive with respect to a differencebetween the RDC1676-01 and RDC1676-00-treated groups; that is, KC showedrelatively little difference between the 2 groups, but the units ofluminosity were very small.

Likewise, FIG. 60 shows analysis of RANTES in BAL data according toparticular exemplary aspects, and showing marked variability in theRDC1676-01 group with one reading being markedly higher than the others,skewing the results.

Likewise, FIG. 61 shows analysis of TNF alpha in BAL data according toparticular exemplary aspects, and showing relatively little significancein the way of difference between the RDC1676-01 and RDC1676-00-treatedgroups.

FIG. 62 shows analysis of MCP-1 in BAL data according to particularexemplary aspects, and showing relatively little significance in the wayof difference between the RDC1676-01 and RDC1676-00-treated groups.

FIGS. 63 through 68 show analysis of MIP1-A, IL-1 alpha, Vcam, IL-1beta, MCP-3, and Eotaxin, respectively, in BAL data according toparticular exemplary aspects, and showing relatively little significancein the way of difference between the RDC1676-01 and RDC1676-00-treatedgroups.

In summary, this standard assay of inflammatory reaction to a knownsensitization produced, at least in the blood samples, a marked clinicaland serologic affect. Additionally, while significant numbers of controlanimals were physiologically stressed and nearly dying in the process,none of the RDC1676-01 treated group showed such clinical stresseffects. This was reflected then in the circulating levels of cytokines,with approximately 30% differences between the RDC1676-01-treated andthe RDC1676-01-treated groups in the OVA challenged groups. By contrast,there were small and fairly insignificant changes in cytokine, cellularand serologic profiles between the RDC1676-01-treated and theRDC1676-01-treated groups in the non-OVA challenged groups, which likelymerely represent minimal baseline changes of the fluid itself.

Example 14 Bradykinin B2 Receptor Affinity Binding

A Bio-Layer Interferometry biosensor, Octet Rapid Extended Detection(RED) (forteBio™) was utilized in order to examine membrane receptoraffinity binding of Bradykinin ligand with the Bradykinin B2 receptor.The biosensor system consists of a polished fiber optic embedded into apolypropylene hub with a sensor-specific chemistry at the tip. Thebiosensor set-up has a layer of molecules attached to the tip of anoptic fiber that creates an interference pattern at the detector. Anychange in the number of molecules bound causes a measured shift in thepattern of light.

As shown in FIG. 69 the Bradykinin B2 membrane receptor was immobilizedonto aminopropylsilane (APS) biosensor. The sample plate set up wasdesignated in FIG. 69 and analyzed in FIG. 70. Next, the binding ofBradykinin to the immobilized receptor was assessed according to thesample set up as designated in FIG. 71. Results of Bradykinin bindingare shown in FIG. 72. Bradykinin binding to the receptor was furthertitrated according to the set-up as designated in FIG. 73.

As indicated in FIG. 74, Bradykinin binding to the B2 receptor wasconcentration dependent, and binding affinity was increased in theproprietary gas-enriched saline fluid of the instant disclosure comparedto normal saline. Stabilization of Bradykinin binding to the B2 receptoris shown in FIG. 75.

Example 15 A Regulatory T-Cell Assay was Used to Show Effects of theInventive Electrokinetically Generated Fluids in Modulation of T-CellProliferation and Elaboration of Cytokines (II-10) and Other Proteins(e.g., GITR, Granzyme A, XCL1, pStat5, and Foxp3)) in Regulatory T-CellAssays, and of, for Example, Tryptase in PBMC

The ability of particular embodiments disclosed herein to regulate Tcells was studied by irradiating antigen presenting cells, andintroducing antigen and T cells. Typically, these stimulated T cellsproliferate. However, upon the introduction of regulatory T cells, theusual T cell proliferation is suppressed.

Methods:

Briefly, FITC-conjugated anti-CD25 (ACT-1) antibody used in sorting waspurchased from DakoCytomation (Chicago, Ill.). The other antibodies usedwere as follows: CD3 (HIT3a for soluble conditions), GITR (PEconjugated), CD4 (Cy-5 and FITC-conjugated), CD25 (APC-conjugated), CD28(CD28.2 clone), CD127-APC, Granzyme A (PE-conjugated), FoxP3(BioLegend), Mouse IgG1 (isotype control), and XCL1 antibodies. Allantibodies were used according to manufacturer's instructions.

CD4+ T cells were isolated from peripheral whole blood with CD4+ RosetteKit (Stemcell Technologies). CD4+ T cells were incubated withanti-CD127-APC, anti-CD25-PE and anti-CD4-FITC antibodies. Cells weresorted by flow cytometry using a FACS Aria into CD4+CD25hiCD127lo/nTregand CD4+CD25− responder T cells.

Suppression assays were performed in round-bottom 96 well microtiterplates. 3.75×103 CD4+CD25neg responder T cells, 3.75×103 autologous Treg, 3.75×104 allogeneic irradiated CD3-depleted PBMC were added asindicated. All wells were supplemented with anti-CD3 (clone HIT3a at 5.0ug/ml). T cells were cultured for 7 days at 37° C. in RPMI 1640 mediumsupplemented with 10% fetal bovine serum. Sixteen hours before the endof the incubation, 1.0 mCi of ³H-thymidine was added to each well.Plates were harvested using a Tomtec cell harvester and ³H-thymidineincorporation determined using a Perkin Elmer scintillation counter.Antigen-presenting cells (APC) consisted of peripheral blood mononuclearcells (PBMC) depleted of T cells using StemSep human CD3+ T celldepletion (StemCell Technologies) followed by 40 Gy of irradiation.

Regulatory T cells were stimulated with anti-CD3 and anti-CD28conditions and then stained with Live/Dead Red viability dye(Invitrogen), and surface markers CD4, CD25, and CD127. Cells were fixedin the Lyze/Fix PhosFlow™ buffer and permeabilized in denaturingPermbuffer III®. Cells were then stained with antibodies against eachparticular selected molecule.

Statistical analysis was performed using the GraphPad Prism software.Comparisons between two groups were made by using the two-tailed,unpaired Student's t-test. Comparisons between three groups were made byusing 1-way ANOVA. P values less than 0.05 were considered significant(two-tailed). Correlation between two groups were determined to bestatistically significant via the Spearman coefficient if the r valuewas greater than 0.7 or less than −0.7 (two-tailed).

Results:

As indicated in FIG. 76, regulatory T cell proliferation was studied bystimulating cells with diesel exhaust particulate matter (PM, from EPA).The x-axis of FIG. 76 shows activated autologous CD4+ effector T cells(responder cells) as a solid black bar, and regulatory T cells alone inthe gray bar (shown for confirmation of anergy) which were mixed at a1:1 ratio as shown in the white bar. The y axis shows proliferation asmeasured by uptake of ³H-thymidine. As shown from left to right alongthe x-axis, “PM” indicates diesel exhaust derived Particulate Matter,“PM+Rev” indicates PM plus a gas-enriched electrokinetically generatedfluid (Rev) of the instant disclosure, “Solis” indicates anelectrokinetically generated fluid of the instant disclosure and devicethat is not gas-enriched beyond ambient atmosphere, only (no PM added),“Rev” indicates Rev alone (no PM added) as defined above, “Media”indicates the cell growth media alone control (minus PM; no Rev, noSolis), and “Saline Con” indicates the saline control (minus PM; no Rev,no Solis), “V” indicates verapamil, and “P” indicates propanolol, and“DT” is DT390 at 1:50.

As shown in FIG. 77, cells stimulated with PM (no Rev, no Solis)resulted in a decrease in secreted IL-10, while cells exposed to PM inthe presence of the fluids of the instant disclosure (“PM+Rev”) resultedin a maintained or only slightly decreased production of IL-10 relativeto the Saline and Media controls (no PM). Furthermore, Diphtheria toxin(DT390, a truncated diphtheria toxin molecule; 1:50 dilution of std.commercial concentration) was titrated into inventive fluid samples, andblocked the Rev-mediated effect of increase in IL-10 in FIG. 77. Notethat treatment with Rev alone resulted in higher IL-10 levels relativeto Saline and Media controls.

Likewise, similar results, shown in FIGS. 78-82, were obtained withGITR, Granzyme A, XCL1, pStat5, and Foxp3, respectively. In Figures,“NSC” is the same as “Solis” (no PM).

FIG. 83 shows AA PBMC data, obtained from an allergic asthma (AA)profile of peripheral blood mononuclear cells (PBMC) evaluatingtryptase. The AA PBMC data was consistent with the above T-regulatorycell data, as cells stimulated with particulate matter (PM) showed highlevels of tryptase, while cells treated with PM in the presence of thefluids of the instant disclosure (“PM+Rev”) resulted in significantlylower tryptase levels similar to those of the Saline and Media controls.Consistent with the data from T-regulatory cells, exposure to DT390blocked the Rev-mediated effect on tryptase levels, resulting in anelevated level of tryptase in the cells as was seen for PM alone (minusRev, no Rev, no Solis). Note that treatment with Rev alone resulted inlower tryptase levels relative to Saline and Media controls.

In summary, the data of FIG. 76, showing a decreased proliferation inthe presence of PM and Rev relative to PM in control fluid (no Rev, noSolis), indicates that the inventive electrokinetically generated fluidRev improved regulatory T-cell function as shown by relatively decreasedproliferation in the assay. Moreover, the evidence of this Example andFIGS. 76-83, indicate that beta blockade, GPCR blockade and Ca channelblockade affects the activity of Revera on Treg function.

Example 16 Treatment of Primary Bronchial Epithelial Cells (BEC) withthe Inventive Electrokinetically Generated Fluids Resulted in ReducedExpression and/or Activity of Two Key Proteins of the AirwayInflammatory Pathways, MMP9 and TSLP

Overview.

As shown in Example 14 above (e.g., FIG. 75, showing Stabilization ofBradykinin binding to the B2 receptor using Bio-Layer Interferometrybiosensor, Octet Rapid Extended Detection (RED) (forteBio™)), Bradykininbinding to the B2 receptor was concentration dependent, and bindingaffinity was increased in the electrokinetically generated fluid (e.g.,Rev; gas-enriched electrokinetically generated fluid) of the instantdisclosure compared to normal saline. Additionally, as shown in Example15 in the context of T-regulatory cells stimulated with diesel exhaustparticulate matter (PM, standard commercial source), the data showed adecreased proliferation of T-regulatory cells in the presence of PM andRev relative to PM in control fluid (no Rev, no Solis) (FIG. 76),indicating that the inventive electrokinetically generated fluid Revimproved regulatory T-cell function; e.g., as shown by relativelydecreased proliferation in the assay. Moreover, exposure to theinventive fluids resulted in a maintained or only slightly decreasedproduction of IL-10 relative to the Saline and Media controls (no PM).Likewise, in the context of the allergic asthma (AA) profiles ofperipheral blood mononuclear cells (PBMC) stimulated with particulatematter (PM), the data showed that exposure to the fluids of the instantdisclosure (“PM+Rev”) resulted in significantly lower tryptase levelssimilar to those of the Saline and Media controls. Additionally, theDiphtheria toxin (DT390, a truncated diphtheria toxin molecule; 1:50dilution of std. commercial concentration) effects shown in Example 15and FIGS. 76-83, indicate that beta blockade, GPCR blockade and Cachannel blockade affects the activity of the electrokineticallygenerated fluids on Treg and PBMC function. Furthermore, the data ofExample 18 shows that, according to additional aspects, upon exposure tothe inventive fluids, tight junction related proteins were upregulatedin lung tissue. FIGS. 85-89 show upregulation of the junction adhesionmolecules JAM 2 and 3, GJA1, 3, 4 and 5 (junctional adherins), OCLN(occludin), claudins (e.g., CLDN 3, 5, 7, 8, 9, 10), TJP1 (tightjunction protein 1), respectively. Furthermore, as shown in the patchclamp studies of Example 23, the inventive electrokinetically generatedfluids (e.g., RNS-60) affect modulation of whole cell conductance (e.g.,under hyperpolarizing conditions) in Bronchial Epithelial Cells (BEC;e.g., Calu-3), and according to additional aspects, modulation of wholecell conductance reflects modulation of ion channels.

In this Example, Applicants have extended these discoveries byconducting additional experiments to measure the effects of productionof two key proteins of the airway inflammatory pathways. Specifically,MMP9 and TSLP were assayed in primary bronchial epithelial cells (BEC).

Materials and Methods:

Commercially available primary human bronchial epithelial cells (BEC)(HBEpC-c from Promocell, Germany) were used for these studies.Approximately 50,000 cells were plated in each well of a 12 well plateuntil they reached ˜80% confluence. The cells were then treated for 6hours with normal saline, control fluid Solas or the test fluid Revera60 at a 1:10 dilution (100 ul in 1 ml of airway epithelial growthmedium) along with the diesel exhaust particulate matter (DEP or PM)before being lifted for FACS analysis, as described in Example 8 herein.Both MMP9 and TSLP receptor antibodies were obtained from BD Biosciencesand used as per manufacturer's specifications.

Results:

In FIGS. 115 and 116, DEP represents cells exposed to diesel exhaustparticulate matter (PM, standard commercial source) alone, “NS”represents cells exposed to normal saline alone, “DEP+NS” representcells treated with particulate matter in the presence of normal saline,“Revera 60” refers to cells exposed only to the test material,“DEP+Revera 60” refer to cells treated with particulate matter in thepresence of the test material Revera 60. In addition, “Solas” and“DEP+Solas” represents cells exposed to the control fluid Solas alone orin combination with the particulate matter, respectively.

FIG. 115 shows that the test material Revera 60 reduces DEP induced TSLPreceptor expression in bronchial epithelial cells (BEC) by approximately90%. Solas resulted in a 55% reduction in TSLP receptor expression,while Normal saline failed to produce similar level of reduction in TSLPreceptor expression (approximately 20% reduction). The effect of theinventive solution in reducing TSLP receptor expression is a significantdiscovery in view of recent findings showing that TSLP plays a pivotalrole in the pathobiology of allergic asthma and local antibody mediatedblockade of TSLP receptor function alleviated allergic disease (Liu, YJ, Thymic stromal lymphopoietin: Master switch for allergicinflammation, J Exp Med 203:269-273, 2006; Al-Shami et al., A role forTSLP in the development of inflammation in an asthma model, J Exp Med202:829-839, 2005; and Shi et al., Local blockade of TSLP receptoralleviated allergic disease by regulating airway dendritic cells, ClinImmunol. Aug. 29, 2008, (Epub ahead of print)).

Likewise, FIG. 116 shows the effect of Revera 60, Solas and normalsaline on the DEP-mediated increase in MMP 9. Specifically, Revera 60inhibited the DEP-induced cell surface bound MMP9 levels in bronchialepithelial cells by approximately 80%, and Solas had an inhibitoryeffect of approximately 70%, whereas normal saline (NS) had a marginaleffect of about 20% reduction. MMP-9 is one of the major proteinasesinvolved in airway inflammation and bronchial remodeling in asthma.Recently, it has been demonstrated that the levels of MMP-9 aresignificantly increased in patients with stable asthma and even higherin acute asthmatic patients compared with healthy control subjects.MMP-9 plays a crucial role in the infiltration of airway inflammatorycells and the induction of airway hyperresponsiveness indicating thatMMP-9 may have an important role in inducing and maintaining asthma(Vignola et al., Sputum metalloproteinase-9/tissue inhibitor ofmetalloproteinase-1 ratio correlates with airflow obstruction in asthmaand chronic bronchitis, Am J Respir Crit Care Med 158:1945-1950, 1998;Hoshino et al., Inhaled corticosteroids decrease subepithelial collagendeposition by modulation of the balance between matrixmetalloproteinase-9 and tissue inhibitor of metalloproteinase-1expression in asthma, J Allergy Clin Immunol 104:356-363, 1999; Simpsonet al., Differential proteolytic enzyme activity in eosinophilic andneutrophilic asthma, Am J Respir Crit Care Med 172:559-565, 2005; Lee etal., A murine model of toluene diisocyanate-induced asthma can betreated with matrix metalloproteinase inhibitor, J Allergy Clin Immunol108:1021-1026, 2001; and Lee et al., Matrix metalloproteinase inhibitorregulates inflammatory cell migration by reducing ICAM-1 and VCAM-1expression in a murine model of toluene diisocyanate-induced asthma, JAllergy Clin Immunol 2003; 111:1278-1284).

According to additional aspects, therefore, the inventiveelectrokinetically generated fluids have substantial therapeutic utilityfor modulating (e.g., reducing) TSLP receptor expression and/or forinhibiting expression and/or activity of MMP-9, including, for example,for treatment of inflammation and asthma.

Example 17 The Inventive Electrokinetically Generated Fluids were Shownto have a Synergistic Anti-Inflammatory Effect with Budesonide in anArt-Recognized Animal Model for Allergic Asthma

This working Example describes experiments performed to assess theairway anti-inflammatory properties of the inventive electrokineticallygenerated fluids (e.g., RDC-1676-03) in a Brown Norway rat ovalbuminsensitization model. The Brown Norway rat is an art-recognized model fordetermining the effects of a test material on airway function and thisstrain has been widely used, for example, as a model of allergic asthma.Airway pathology and biochemical changes induced by ovalbuminsensitization in this model resemble those observed in man (Elwood etal., J Allergy Clin Immuno 88:951-60, 1991; Sirois & Bissonnette, ClinExp Immunol 126:9-15, 2001). The inhaled route was selected to maximizelung exposure to the test material or the control solution. Theovalbumin-sensitized animals were treated with budesonide alone or incombination with the test material RDC 1676-03 for 7 days prior toovalbumin challenge. 6 and 24 hours following the challenge, total bloodcount and levels of several pro and anti-inflammatory cytokines as wellas various respiratory parameters were measured to estimate anybeneficial effect of administering the test material on variousinflammatory parameters.

Materials and Methods:

Brown Norway rats of strain Bn/Crl were obtained from Charles RiverKingston, weighing approximately 275±50 g at the onset of theexperiment. All animal studies were conducted with the approval byPCS-MTL Institutional Animal Care and Use Committee. During the study,the use and care of animals were conducted according to guidelines ofthe USA National Research Council as well as Canadian Council of AnimalCare.

Sensitization.

On day 1 of the experiment, animals (14 animals in each treatment group)were sensitized by administration of a 1 ml intraperitoneal injection ofa freshly prepared solution of 2 mg ovalbumin/100 mg Aluminum Hydroxideper 1 ml of 0.9% Sodium Chloride, followed by repeat injection on day 3.

Treatment.

Fifteen days following the initial sensitization, animals were subjectedto nebulized exposure to control (Normal saline) or test solutions(electrokinetically generated fluids RDC1676-00, RDC1676-02 andRDC-1676-03), either administered alone or in combination withBudesonide, once daily for 15 minutes for 7 consecutive days. Animalswere dosed in a whole body chamber of approximately 20 L, and testatmosphere was generated into the chamber air inlet using aeronebultrasonic nebulizers supplied with air from a Buxco bias flow pump. Theairflow rate was set at 10 liters/min.

Ovalbumin Challenge.

On day 21, 2 hours following treatment with the test solutions, allanimals were challenged with 1% ovalbumin nebulized solution for 15minutes (in a whole body chamber at airflow 2 L/min).

Sample Collection.

At time points of 6 and 24 hours after the ovalbumin challenge, bloodsamples were collected for total and differential blood cell counts aswell as for measuring levels of various pro and anti-inflammatorycytokines. In addition, Immediately after and at 6 and 24 hoursfollowing ovalbumin challenge the enhanced pause Penh and tidal volumewere measured for a period of 10 minutes using the Buxco ElectronicsBioSystem XA system.

Results:

Eosinophil Count: As expected, and shown in FIG. 109, treatment withBudesonide (“NS+Budesonide 750 μg/Kg”; densely crosshatched bar graph)reduced the total eosinophil count in the challenged animals relative totreatment with the normal saline “NS” alone control (open bar graph).Additionally, while treatment with the inventive fluid “RDC1676-03”alone (lightly crosshatched bar graph) did not significantly reduce theeosinophil count, it nonetheless displayed a substantial synergy withBudesonide in reducing the eosinophil count (“RDC1676-03+Budesonide 750μg/Kg”, solid dark bar graph). Similarly, in FIG. 110, the Eosinophil %also reflected a similar trend. While RDC1676-03 (lightly crosshatchedgraph bar) or Budesonide 750 ug/kg (densely crosshatched bar graph)alone did not have a significant effect on Eosinophil % count in thechallenged animals, the two in combination reduced the Eosinophil %significantly (solid dark bar graph).

Therefore, FIGS. 109 and 110 show, according to particular aspects ofthe present invention that the inventive electrokinetically generatedfluids (e.g., RDC1676-03) were demonstrated to have a substantialsynergistic utility in combination with Budesonide to significantlyreduce eosinophil count (“Eosinophil %” and total count) in anart-recognized rat model for human allergic asthma.

Respiratory Parameters:

FIGS. 111A-C and 112A-C demonstrate the observed effect of the testfluids on Penh and tidal volume as measured immediately, 6 and 24 hoursafter the ovalbumin challenge. Penh is a derived value obtained frompeak inspiratory flow, peak expiratory flow and time of expiration andlowering of penh value reflects a favorable outcome for lung function.Penh=(Peak expiratory flow/Peak inspiratory flow)*(Expiratory time/timeto expire 65% of expiratory volume−1).

As evident from FIGS. 111A-C, treatment with Budesonide (at both 500 and750 ug/kg) alone or in combination with any of the test fluids failed tosignificantly affect the Penh values immediately after the challenge.However, 6 hours after the challenge, animals treated with RDC1676-03alone or in combination with Budesonide 500 or 750 ug/kg demonstrated asignificant drop in Penh values. Although the extent of this drop wasdiminished by 24 hours post challenge, the trend of a synergistic effectof Budesonide and RDC fluid was still observed at this time point.

Tidal volume is the volume of air drawn into the lungs duringinspiration from the end-expiratory position, which leaves the lungspassively during expiration in the course of quiet breathing. As shownin FIGS. 112A-C, animals treated with Budesonide alone showed no changein tidal volumes immediately after the challenge. However, RDC1676-03alone had a significant stimulatory effect on tidal volume even at thisearly time point. And again, RDC1676-03 in combination with Budesonide(both 500 and 750 ug/kg) had an even more pronounced effect on Tidalvolume measurements at this time point. Six hours after the challenge,RDC1676-03 alone was sufficient to cause a significant increase in tidalvolume and addition of Budesonide to the treatment regimen either aloneor in combination had no added effect on tidal volume. Any effectobserved at these earlier time points were, however, lost by the 24hours time point.

Taken together, these data demonstrate that RDC1676-03 alone or incombination with Budesonide provided significant relief to airwayinflammation as evidenced by increase in tidal volume and decrease inPenh values at 6 hours post challenge.

Cytokine Analysis:

To analyze the mechanism of the effects seen on the above discussedphysiological parameters, a number of pro as well as anti-inflammatorycytokines were measured in blood samples collected at 6 and 24 hoursafter the challenge, immediately following the physiologicalmeasurements.

FIGS. 113A and 113B clearly demonstrate that Rev 60 (or RDC1676-03)alone lowered the blood level of eotaxin significantly at both 6 and 24hours post challenge. Budesonide 750 ug/kg also reduced the bloodeotaxin levels at both of these time points, while Budesonide 250 ug/kgonly had a notable effect at the later time point. However, the testsolution Rev 60 alone showed effects that are significantly more potent(in reducing blood eotaxin levels) than both concentrations ofBudesonide, at both time points. Eotaxin is a small C-C chemokine knownto accumulate in and attract eosinophils to asthmatic lungs and othertissues in allergic reactions (e.g., gut in Crohn's disease). Eotaxinbinds to a G protein coupled receptor CCR3. CCR3 is expressed by anumber of cell types such as Th2 lymphocytes, basophils and mast cellsbut expression of this receptor by Th2 lymphocyte is of particularinterest as these cells regulate eosinophil recruitment. Several studieshave demonstrated increased production of eotaxin and CCR3 in asthmaticlung as well as establishing a link between these molecules and airwayhyperresponsiveness (reviewed in Eotaxin and the attraction ofeosinophils to the asthmatic lung, Dolores M Conroy and Timothy JWilliams Respiratory Research 2001, 2:150-156). It is of particularinterest to note that these studies completely agree with the results inFIGS. 109 and 110 on eosinophil counts.

Taken together these results strongly indicate that treatment withRDC1676-03 alone or in combination with Budesonide can significantlyreduce eosinophil total count and % in blood 24 hours after theovalbumin challenge. This correlates with a significant drop in eotaxinlevels in blood observed as early as 6 hours post challenge.

Blood levels of two major key anti-inflammatory cytokines, IL10 andInterferon gamma are also significantly enhanced at 6 hours afterchallenge as a result of treatment with Rev 60 alone or in combinationwith Budesonide. FIGS. 113C and 113D show such effects on Interferongamma and IL 10, respectively. It is evident from these figures that Rev60 alone or Rev 60 in combination with Budesonide 250 ug/kgsignificantly increased the blood level of IL10 in the challengedanimals up to 6 hrs post challenge. Similarly, Rev 60 alone or incombination with Budesonide 250 or 750 ug/kg significantly increased theblood level of IFN gamma at 6 hours post challenge. Increase in theseanti-inflammatory cytokines may well explain, at least in part, thebeneficial effects seen on physiological respiratory parameters seen 6hours post challenge. The effect on these cytokines was no longerobserved at 24 hour post challenge (data not shown).

Rantes or CCL5 is a cytokine expressed by circulating T cells and ischemotactic for T cells, eosinophils and basophils and has an activerole in recruiting leukocytes into inflammatory sites. Rantes alsoactivates eosinophils to release, for example, eosinophilic cationicprotein. It changes the density of eosinophils and makes them hypodense,which is thought to represent a state of generalized cell activation. Italso is a potent activator of oxidative metabolism specific foreosinophils.

As shown in FIG. 114, systemic levels of Rantes was reducedsignificantly at 6 hours, but not at 24 hours post challenge in animalstreated with Rev 60 alone or in combination of Budesonide 250 or 750ug/kg. Once again, there is a clear synergistic effect of Budesonide 750ug/kg and Rev 60 that is noted in this set of data. A similar downwardtrend was observed for a number of other pro-inflammatory cytokines,such as KC or IL8, MCP3, IL1b, GCSF, TGFb as well as NGF, observedeither at 6 or at 24 hours post challenge, in animals treated with Rev60alone or in combination with Budesonide.

Example 18 The Inventive Therapeutic Fluids Have Substantial Utility forModulating Intercellular Light Junctions

According to particular aspects, the inventive diffuser processedtherapeutic fluids have substantial utility for modulating intercellulartight junctions, including those relating with pulmonary and systemicdelivery and bioavailability of polypeptides, including the exemplarypolypeptide salmon calcitonin (sCT).

Example Overview.

Salmon calcitonin (sCT) is a 32 amino acid peptide with a molecularweight of 3,432 Daltons. Pulmonary delivery of calcitonin has beenextensively studied in model systems (e.g., rodent model systems, ratmodel systems, etc) to investigate methods to enhance pulmonary drugdelivery (e.g., intratracheal drug delivery). According to particularexemplary aspects, the inventive diffuser processed therapeutic fluidhas substantial utility for modulating (e.g., enhancing) intercellulartight junctions, for example those associated with pulmonary andsystemic delivery and bioavailability of sCT in a rat model system.

Methods:

Intratracheal Drug Delivery.

According to particular embodiments, sCT is formulated in the inventivetherapeutic fluid and administered to rats using an intratracheal drugdelivery device. In certain aspects, a Penn Century Micro-Sprayer devicedesigned for rodent intratracheal drug delivery is used, allowing forgood lung delivery, but, as appreciated in the art, with relatively lowalveolar deposition resulting in poor systemic bioavailability ofpeptides. According to particular aspects, this art-recognized modelsystem was used to confirm that the inventive diffuser processedtherapeutic fluid has substantial utility for modulating (e.g.,enhancing) intercellular tight junctions, including those associatedwith pulmonary and systemic delivery and bioavailability ofpolypeptides.

Animal Groups and Dosing.

In certain aspects, rats are assigned to one of 3 groups (n=6 pergroup): a) sterile saline; b) base solution without O₂ enrichment (‘basesolution’); or c) inventive diffuser processed therapeutic fluid(‘inventive enriched based solution’). The inventive enriched basedsolution is formed, for example by infusing oxygen in 0.9% saline.Preferably, the base solution comprises about 0.9% saline to minimizethe potential for hypo-osmotic disruption of epithelial cells. Incertain embodiments, sCT is separately reconstituted in the basesolution and the inventive enriched based solution and the respectivesolutions are delivered to respective animal groups by intratrachealinstillation within 60 minutes (10 μg sCT in 200 μL per animal).

Assays.

In particular aspects, blood samples (e.g., 200 μl) are collected andplaced into EDTA coated tubes prior to dosing and at 5, 10, 20, 30, 60,120 and 240 minutes following dosing. Plasma is harvested and stored at≦−70° C. until assayed for sCT using an ELISA.

For Agilant gene array data generation, lung tissue was isolated andsubmerged in TRI Reagent (TR118, Molecular Research Center, Inc.).Briefly, approximately 1 mL of TRI Reagent was added to 50-100 mg oftissue in each tube. The samples were homogenized in TRI Reagent, usingglass-Teflon™ or Polytron™ homogenizer. Samples were stored at −80° C.

Results:

Enhancement of Tight Junctions.

FIG. 84 shows that RDC1676-01 (sterile saline processed through theinstant proprietary device with additional oxygen added; gas-enrichedelectrokinetically generated fluid (Rev) of the instant disclosure)decreased systemic delivery and bioavailability of sCT. According toparticular aspects, the decreased systemic delivery results fromdecreased adsorption of sCT, most likely resulting from enhancement ofpulmonary tight junctions. RDC1676-00 signifies sterile saline processedaccording to the presently disclosed methods, but without oxygenation.

Additionally, according to particular aspects, tight junction relatedproteins were upregulated in lung tissue. FIGS. 85-89 show upregulationof the junction adhesion molecules JAM 2 and 3, GJA1, 3, 4 and 5(junctional adherins), OCLN (occludin), claudins (e.g., CLDN 3, 5, 7, 8,9, 10), TJP1 (tight junction protein 1), respectively.

Example 19 The Inventive Therapeutic Fluids have Substantial Utility forModulating Nitric Oxide Levels

According to particular aspects, the inventive diffuser processedtherapeutic fluids have substantial utility for modulating nitric oxidelevels, and/or related enzymes. FIGS. 90-94 show data obtained fromhuman foreskin keratinocytes exposed to RDC1676-01 (sterile salineprocessed through the instant proprietary device with additional oxygenadded; gas-enriched electrokinetically generated fluid (Rev) of theinstant disclosure) showing up-regulation of NOS1 and 3, and Nostrin,NOS3. By contrast, data obtained from rat lung tissue (tissue of aboveExample entitled “Cytokine Expression”) shows down regulation of NOS2and 3, Nostrin and NOS1AP with Rev (FIGS. 93, 94).

Example 20 Localized Electrokinetic Effects (Voltage/Current) wereDemonstrated Using a Specially Designed Mixing Device ComprisingInsulated Rotor and Stator Features

In this Example, feature-localized electrokinetic effects(voltage/current) were demonstrated using a specially designed mixingdevice comprising insulated rotor and stator features.

Overview.

As discussed in detail herein above under “Double Layer Effect” (seealso FIGS. 26 and 28) The mixing device 100 may be configured to createthe output material 102 by complex and non-linear fluid dynamicinteraction of the first material 110 and the second material 120 withcomplex, dynamic turbulence providing complex mixing that further favorselectrokinetic effects. According to particular aspects, the result ofthese electrokinetic effects may be present within the output material102 as charge redistributions and redox reactions, including in the formof solublized electrons that are stabilized within the output material.

In addition to general surface-related double layer effects in themixing chamber, Applicants additionally reasoned that localizedelectrokinetic effects may be imparted by virtue of the feature-inducedmicrocavitation and fluid acceleration and deceleration in the vicinityof the features. The studies of this Example were thus performed tofurther investigate and confirm said additional electrokinetic aspects.

Materials:

A test device similar to the inventive mixing devices described hereinwas constructed, comprising a stainless steel rotor 12 having twofeatures 18 (disposed at 180 degrees), and a stator 14 with a singlefeature 16 positioned to be rotationally opposable to the rotor features18 and stator features 16. Significantly, the rotor and stator features,in each case, are insulated from the respective rotor and stator bodies(FIG. 95). The device was machined to provide for a consistentrotor:stator gap 20 of 0.020 inches to conform with the devicesdisclosed elsewhere herein. There is a rotating contact (not shown) atthe end of the rotor shaft (not shown) that provides an electrical pathfor the rotor surface and for the insulated rotor features. Likewise thestator has a similar insulated feature 16 (FIG. 95), wherein the statorinner surface and the insulated stainless steel feature are connected torespective contacts on the stator exterior.

A operational amplifier (OpAmp) circuit (M) 22 is connected between thecontacts. The operational amplifier (OpAmp) circuit was constructed toprovide for collection of very low voltage measurements by takingadvantage of the high input impedance of such amplifiers. The outputs ofthe OpAmp are fed to the inputs of an oscilloscope (e.g., a batterypowered laptop running an oscilloscope application with a Pico Scope3000™).

To eliminate the introduction of any ambient noise (e.g., RF radiationfrom wireless network signals and from the 60 Hz power line) duringtesting of the device, a fine copper mesh, RF-shielded compartment(approx. three by four by four feet) was constructed to provide aFaraday cage. This configuration provided for excellent signal to noiseratios during experimental testing, as interfering signals from 60 Hz ACnoise (e.g., of approximately two volts) and high frequency RF wasreduced well below the signals of interest. Using a battery poweredlaptop running an oscilloscope application with a Pico Scope 3000enabled detection of the 30 mV signals (as in FIG. 96) created by thefeatures of the test device. In addition, a variable speed DC motor waspositioned outside the Faraday cage and coupled to the rotatable testdevice via a non-metallic shaft to effectively isolate the motor noiseaway from the test device.

Methods:

The OpAmp circuit was used to measure voltage potential between thecontacts connecting the stator inner surface 12 and the insulated statorfeature 16. With the particular circuit arrangement, only a potentialwas measured. The rotational speed of the device could be varied betweenabout 700 to about 2800 rpm (with the data of FIG. 96 being measuredwith the device running at about 1800 rpm).

To avoid any extraneous voltage generation due to a pump or peristalticpump, fluid flow through the device was accomplished using inertnitrogen or air or argon acting on fluid in tanks connected to thedevice. There was no perceptible voltage contribution from the flowmechanism, and typically air was used as the pumping force to providefor fluid flow through the device.

Fluid flow rate through the device was about 1 L/min.

An initial set of non-rotational experiments was conducted by directingfluid flow through the device chamber but without rotation of the rotorin order to assess the presence of any voltage between the stator body12 and the isolated feature 16. Separate experiments were conducted forboth flow directions.

An additional set of rotational experiments was then conducted with thesame fluid flow rate, and with the device rotor rotating at variousspeeds from about 300 to about 1800 rpm. For any given experiment, theflow rate and rotational speed were held constant.

Results:

With respect to the non-rotational experiments, with fluid flowingthrough the device in either direction without any rotor rotation therewas only a barely perceptible voltage (e.g., 1 to 2 mV)) between thebody of the stator and the insulated feature.

With respect to the rotational experiments, and with reference to FIG.96, it can be seen that voltage pulses (potential pulses), temporallycorrelating (in this case at about 1800 rpm) with rotational alignmentof opposing rotor stator features, were measurable with the OpAmp in theoperating test device. Moreover, such periodic voltage pulses,correlating with feature alignments, could be observed over a range fromabout 250 or 300 rpm to about 1800. Additionally, with or without fluidflow, such voltage pulses were observed in the rotational experiments aslong as the cavity/fluid chamber of the device was filled with fluid.According to particular aspects, and without being bound by mechanism,rapid, violent compression (e.g., cavitation), acceleration anddeceleration of fluid flow in the vicinity of the repetitiverotationally aligned features created the respective local voltagepulses that correlate exactly with the rotational period, providing, atleast in part, for electrokinetically generated fluid according to thepresent invention. Additional experiments revealed that the amplitude(peak shape and height) of the voltage pulses increased with increasingrotational velocity, being initially observable at about 250 to 300 rpmin this particular test device, and increasing up to at least about 2800rpm. The magnitude of the violent acceleration and deceleration, etc.,of fluid flow in the vicinity of the rotationally aligned features wouldbe expected to generally increase with increasing rotational velocity;at least until a maximum was reached reflecting physical limits imposedby the geometry, configuration and/or flow rate of the device. Accordingto additional aspects, because localized voltage spikes are present,localized current flow (e.g., current pulses) is generated in thevicinity of the features, providing, at least in part, forelectrokinetically generated fluid according to the present invention(e.g., without being bound by mechanism, providing for electrochemicalreactions as discussed elsewhere herein).

According to additional aspects, and without being bound by mechanism,such feature-localized effects (e.g., voltage pulses and current and/orcurrents pulses) contribute to generation of the electrokineticallygenerated fluids in combination with more general surface-related doublelayer and streaming current effects discussed elsewhere herein aboveunder “Double Layer Effect” (see also FIGS. 26 and 28).

Example 21 Relative to Non-Electrokinetically Generated Control Fluids,the Inventive Electrokinetically Generated Fluids Were Shown toDifferentially Affect Line Widths in ¹³C NMR Analysis of the DissolvedSolute α,α-Trehalose

Overview.

Applicants data disclosed elsewhere herein support utility and mechanismwherein the inventive electrokinetically generated fluids mediateregulation or modulation of intracellular signal transduction bymodulation of at least one of cellular membranes, membranepotential/conductance, membrane proteins (e.g., membrane receptors suchas G protein coupled receptors), calcium dependant cellular signalingsystems, and intercellular junctions (e.g., tight junctions, gapjunctions, zona adherins and desmasomes). Specifically, using a varietyof art-recognized biological test systems and assays, Applicants datashows, relative to control fluids, differential effects of the inventivefluid on, for example: regulatory T cell proliferation; cytokine andprotein levels (e.g, IL-10, GITR, Granzyme A, XCL1, pStat5, and Foxp3,tyrptase, tight junction related proteins, TSLP receptor, MMP9, etc.);binding of Bradykinin ligand with the Bradykinin B2 receptor; expressionof TSLP receptor, whole cell conductance; etc. Moreover, the Diphtheriatoxin (DT390) effects shown herein indicate that beta blockade (beta 2adrenergic receptor), and/or GPCR blockade and/or Ca channel blockadeaffects the activity of the electrokinetically generated fluids on, forexample, Treg and PBMC function.

Taken together these effects indicate that the inventiveelectrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids, but also that they provide fornovel compositions and substantial utilities such as those presentlydisclosed and claimed herein.

In this Example.

Applicants have in this Example performed nuclear magnetic resonance(NMR) studies to further characterize the fundamental nature of theinventive electrokinetically generated fluids. Specifically, Applicantshave analyzed the ¹³C NMR spectra of α,α-Trehalose dissolved in theelectrokinetically generated fluid, compared to dissolution innon-electrokinetically generated fluid. Trehalose (shown below withcarbons numbered for reference) is a cosmotrophic solute and is known,for example to protect against protein denaturation, membranedesiccation, organism viability upon freezing, etc. Applicants, giventhe data summarized above, reasoned that α,α-Trehalose might provide aneffective tool to further probe the properties/structure of theinventive electrokinetically generated fluids. Applicants reasoned thatNMR-related ‘chemical shifts’ and effects on ‘line widths’ could be usedto assess properties of the inventive fluids. For these studies, anon-superoxygenated inventive electrokinetically generated fluid(referred to herein as “Solas”) was employed to minimize the possibilitythat paramagnetic impurities, such as dissolved oxygen, might act tocounter or otherwise mask the effects being analyzed.

Materials and Methods:

Solution Preparation.

The Phosphate (sodium salt) and D-(+)-Trehalose dihydrate (T9531-10G,reduced metal content) and 99.9% D2O containing 1% DSS were purchasedfrom Sigma. The “Normal Saline” is 0.9% Sodium Chloride, pH 5.6(4.5-7.0), from Hospira. The 0.25 M α,α-Trehalose solutions wereprepared by dissolving 0.949 g trehalose into 965 μL Normal Saline and35 mL Phoshate Buffered Saline (100 mM Phosphate Buffer in 0.9% NaClpreparted in such a way that when 35 μL of this buffer are added to 1.0mL trehalose solution the pH becomes 6.93).

Nuclear Magnetic Resonance Spectra Collection.

Spectra were collected at the University of Washington NMR facilityusing either an 500 MHz or 300 MHz Bruker Avance series instrumentfitted with a Bruker BBO: X {1H} probe and running XWINNMR 3.5. ¹³C NMRspectra were collected at 125.7 MHz or 75.46 MHz using a 14000 Hz or7900 Hz sweep width using 64K or 128K data points and 128 or 256 scans.The resulting FIDs were zero-filled twice and processed with a 1.0 Hzline broadening factor. Temperature was controlled using the BrukerBiospin Variable Temperature unit. External deuterium locking wasemployed by placing 99.9% D2O+1% DSS+ a trace of acetone in a coaxialNMR insert tube, purchased from Wilmad. The NMR data was processed usingthe iNMR software v. 2.6.4 from Mestrelab Research.

Results:

Sample Spectra.

FIG. 97A-C shows expansions of six ¹³C-NMR spectra overlaid on top ofeach other such that the DSS signals line up at −2.04 ppm. The DSSsignals are shown at the far right of the figure, and the acetone methylsignal is shown near 30.9 ppm. The remaining signals correspond to the 6carbons of trehalose as shown in the α,α-Trehalose structure above. Ascan be seen, the carbon signals in the Solas solutions show smallchemical shifts (generally upfield) compared to the control solutions.

Line Width Measurements.

TABLE 9 below shows the measured ¹³C NMR line widths for the six carbonsof trehalose and the methyl carbon of acetone at 3 differenttemperatures for Solas Saline (an inventive electrokinetically generatedfluid). The corresponding Normal Saline samples representnon-electrokinetic control solutions at each temperature. In the Solassolutions, the line widths are significantly different from the linewidths in the control solution for each carbon atom. The smallerlinewidths in the Solas solutions at lower temperatures likely resultfrom a faster tumbling rate of the trehalose molecule as a whole(including any solvated water molecules) compared to the controlsolutions.

TABLE 9 ¹³C NMR Line Widths for α,α-Trehalose in Solas & NormalSaline^(a,b) Test Fluid (Temp. degrees K) C-1 C-2 C-3 C-4 C-5 C-6Acetone Solas (277) 8.4 8.22 8.3 8.15 8.3 11.1 5.1 Normal (269.9) 15.416.1 15.8 14.9 15.4 21.7 5.1 Solas (293) 9.52 8.7 9.28 9 8.9 11.25 5.63Normal (292.9) 10.33 10.23 10.23 9.93 10.23 13.13 5.63 Solas (310) 2.282.03 2.18 2.19 2 2.55 0.67 Normal (309.9) 1.17 0.99 1.1 1.02 0.97 1.420.67 ^(a)1.0 Hz was subtracted from all line width values due to the 1.0Hz line broadening used during processing. In addition, line widthvalues were normalized relative to the acetone signal in the externalreference tube in order to compensate for magnetic fieldinhomogeneities. This was done by subtracting from the Normal Salineline widths the amount by which the acetone peak was broadened in thecorresponding Solas Saline spectra. ^(b)Error in line width measurementsestimated to be within +/−0.30 Hz

The ¹³C NMR line widths for □.□-Trehalose in Solas and normal saline, ineach case normalized with respect to the Acetone line, are showngraphically in FIG. 97A. In conclusion, the NMR data for ¹³C NMR linewidths for α,α-Trehalose in Solas and normal saline indicate that thereis a property of the inventive solution which alters solute tumbling.

Taken together with the biological activities summarize above andelsewhere herein, these ¹³C NMR line width effects indicate that theinventive electrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids in terms of solute interactions, butalso that they provide for novel compositions and substantial utilitiessuch as those presently disclosed and claimed herein.

Example 22 Relative to Non-Electrokinetically Generated Control Fluids,the Inventive Electrokinetically Generated Fluids Produced DifferentialSquare Wave Voltametry Profiles and Displayed Unique ElectrochemicalProperties Under Stripping Polarography

Overview.

Applicants' data disclosed elsewhere herein support utility andmechanism wherein the inventive electrokinetically generated fluidsmediate regulation or modulation of intracellular signal transduction bymodulation of at least one of cellular membranes, membranepotential/conductance, membrane proteins (e.g., membrane receptors suchas G protein coupled receptors), calcium dependant cellular signalingsystems, and intercellular junctions (e.g., tight junctions, gapjunctions, zona adherins and desmasomes). Specifically, using a varietyof art-recognized biological test systems and assays. Applicants datashows, relative to control fluids, differential effects of the inventivefluid on, for example: regulatory T cell proliferation; cytokine andprotein levels (e.g, IL-10, GITR, Granzyme A, XCL1, pStat5, and Foxp3,tyrptase, tight junction related proteins, TSLP receptor, MMP9, etc.);binding of Bradykinin ligand with the Bradykinin B2 receptor; expressionof TSLP receptor, whole cell conductance; etc. Moreover, the Diphtheriatoxin (DT390) effects shown herein indicate that beta blockade (beta 2adrenergic receptor), and/or GPCR blockade and/or Ca channel blockadeaffects the activity of the electrokinetically generated fluids on, forexample, Treg and PBMC function.

Taken together these effects indicate that the inventiveelectrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids, but also that they provide fornovel compositions and substantial utilities such as those presentlydisclosed and claimed herein.

In this Example.

Applicants have, in this Example, performed voltametry studies tofurther characterize the fundamental nature of the inventiveelectrokinetically generated fluids. Voltametry is frequently used todetermine the redox potential or measure kinetic rates and constants offluids. The common characteristic of all voltametric methods is thatthey involve the application of a potential to an electrode and theresultant current flowing is monitored through an electrochemical cell.The applied potential produces a change in the concentration of anelectroactive species at the electrode surface by electrochemicallyreducing or oxidizing the species.

Specifically, Applicants have utilized voltametric methods (i.e., squarewave voltametry and stripping polarography) to further characterizefundamental differences between control saline fluid and the inventiveelectrokinetically generated test fluids (e.g., Solas and Revera).Applicants, given the biological and membrane effects data summarizedabove, reasoned that square wave voltametry and stripping polarographywould provide an effective means to further characterize the uniqueproperties of the inventive electrokinetically generated fluids.

Applicants further reasoned that differences in current at specificvoltages, production of different concentrations of an electroactiveredox compound, creation of new redox compounds, and possession ofunique electrochemical properties could be used to assess andcharacterize properties of the inventive fluids. For these studies, botha superoxygenated electrokinetically generated fluid (Revera), and anon-superoxygenated inventive electrokinetically generated fluid (Solas)were used.

Materials and Methods:

Materials and Solution Preparation.

The experiments were conducted on an EG & G SMDE 303A polarographer(Princeton Applied Research). The electrolyte, NaOH, used in the squarewave voltametry experiment, was purchased from Sigma. A 10 mL sample ofthe inventive fluid solution was prepared by adding 100 μL of NaOH to9.9 mL of Revera Saline to make a 0.18 molar solution. With regards tothe stripping polarography experiment, no extra electrolyte wasutilized.

Square Wave Voltametry.

As stated above, voltametry is used to determine the redox potential ormeasure kinetic rates and constants in fluids. In the square wavevoltametry experiment, a potential of 0.0 to approximately −1.75 V wasapplied to an electrode and the resultant current flowing through theelectrochemical cell was monitored.

Stripping Polarography.

The stripping polarography method is similar to the square wavevoltametry method. However, no electrolyte was utilized as stated aboveand also involved a pre-step. In the pre-step, the static mercury dropelectrode was held for 30 seconds at −1.1 V to amalgamate any compoundswhose reduced form was soluble in mercury. Then, the potentials between−1.1 V and 0.0 V were scanned and the resultant current flowing throughthe electrochemical cell was monitored. A linear scan into the negativepotentials on this amalgam provided a sensitive measurement of thesecompounds.

Results:

Square Wave Voltametry.

As evident from FIG. 98, the current profiles at −0.14V, −0.47V, −1.02Vand −1.36V differ between the various tested agents. According toparticular aspects, the differences in current generated at the variousspecific voltages indicate at least one of a different concentration ofan electroactive redox compound and/or a new or unique electroactiveredox compound, and/or a change in the diffusion-limiting electricaldouble layer surrounding the mercury drop.

Stripping Polarography.

FIG. 99 shows that the inventive electrokinetically generated fluids,Revera and Solas, show unique spectra with pronounced peaks at −0.9volts that are not present in the non-electrokinetically generated blankand saline control fluids. Additionally, the spectra of thenon-electrokinetically generated blank and saline control fluids showcharacteristic peaks at −0.19 and −0.3 volts that are absent in thespectra for the electrokinetically generated Solas and Revera fluids.

According to particular aspects, therefore, these results show uniqueelectrochemical properties of the inventive electrokinetically generatedSolas and Revera fluids compared to non-electrokinetically generatedSaline control fluid. According to additional aspects, the resultsindicate the presence or generation of at least one of a differentconcentration of an electroactive redox compound and a new and/or uniqueelectroactive redox compound in electrokinetically generated versusnon-electrokinetically generated fluids.

On top of the various biological data presented elsewhere herein, thisdifferential voltametry data, particularly when considered along withthe differential effects on whole cell conductance, ¹³C NMR line-widthanalysis, and the mixing device feature-localized effects (e.g., voltagepulses and current and/or currents pulses) indicate that the inventiveelectrokinetically generated fluids are not only fundamentallydistinguished from prior art fluids, but also provide for novelcompositions and substantial utilities such as those presently disclosedand claimed herein.

Example 23 Patch Clamp Analysis Conducted on Bronchial Epithilial Cells(BEC) Perfused with Inventive Electrokinetically Generated Fluid(RNS-60) Revealed that Exposure to RNS-60 Resulted in a Decrease inWhole Cell Conductance, and Stimulation with a cAMP Stimulating“Cocktail”, which Dramatically Increased the Whole-Cell Conductance, andAlso Increased the Drug-Sensitive Portion of the Whole-Cell Conductance,which was Ten-Times Higher than that Observed Under Basal Conditions

In this Example, patch clamp studies were performed to further confirmthe utility of the inventive electrokinetically generated fluids tomodulate intracellular signal transduction by modulation of at least oneof membrane structure, membrane potential or membrane conductivity,membrane proteins or receptors, ion channels, and calcium dependantcellular messaging systems.

Overview.

As shown in Example 14 above (e.g., FIG. 75, showing Stabilization ofBradykinin binding to the B2 receptor using Bio-Layer Interferometrybiosensor, Octet Rapid Extended Detection (RED) (forteBio™)), Bradykininbinding to the B2 receptor was concentration dependent, and bindingaffinity was increased in the electrokinetically generated fluid (e.g.,Rev; gas-enriched electrokinetically generated fluid) of the instantdisclosure compared to normal saline. Additionally, as shown in Example15 in the context of T-regulatory cells stimulated with particulatematter (PM), the data showed a decreased proliferation of T-regulatorycells in the presence of PM and Rev relative to PM in control fluid (noRev, no Solis) (FIG. 76), indicating that the inventiveelectrokinetically generated fluid Rev improved regulatory T-cellfunction; e.g., as shown by relatively decreased proliferation in theassay. Moreover, exposure to the inventive fluids resulted in amaintained or only slightly decreased production of IL-10 relative tothe Saline and Media controls (no PM). Likewise, in the context of theallergic asthma (AA) profiles of peripheral blood mononuclear cells(PBMC) stimulated with particulate matter (PM), the data showed thatexposure to the fluids of the instant disclosure (“PM+Rev”) resulted insignificantly lower tryptase levels similar to those of the Saline andMedia controls. Additionally, the Diphtheria toxin (DT390) effects shownin Example 15 and FIGS. 76-83, indicate that beta blockade, GPCRblockade and Ca channel blockade affects the activity of theelectrokinetically generated fluids on Treg and PBMC function.Furthermore, the data of Example 18 shows that, according to additionalaspects, upon expose to the inventive fluids, tight junction relatedproteins were upregulated in lung tissue. FIGS. 85-89 show upregulationof the junction adhesion molecules JAM 2 and 3, GJA1, 3, 4 and 5(junctional adherins), OCLN (occludin), claudins (e.g., CLDN 3, 5, 7, 8,9, 10), TJP1 (tight junction protein 1), respectively.

Patch clamp studies were performed to further investigate and confirmsaid utilities.

Materials and Methods:

The Bronchial Epithelial line Calu-3 was used in Patch clamp studies.Calu-3 Bronchial Epithelial cells (ATCC #HTB-55) were grown in a 1:1mixture of Ham's F12 and DMEM medium that was supplemented with 10% FBSonto glass coverslips until the time of the experiments. In brief, awhole cell voltage clamp device was used to measure effects on Calu-3cells exposed to the inventive electrokinetically generated fluids(e.g., RNS-60; electrokinetically treated normal saline comprising 60ppm dissolved oxygen; sometimes referred to as “drug” in this Example).

Patch clamping techniques were utilized to assess the effects of thetest material (RNS-60) on epithelial cell membrane polarity and ionchannel activity. Specifically, whole cell voltage clamp was performedupon the Bronchial Epithelial line Calu-3 in a bathing solutionconsisting of: 135 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, and 10mM HEPES (pH adjusted to 7.4 with N-methyl D-Glucamine). Basal currentswere measured after which RNS-60 was perfused onto the cells.

More specifically, patch pipettes were pulled from borosilicate glass(Garner Glass Co, Claremont, Calif.) with a two-stage Narishige PB-7vertical puller and then fire-polished to a resistance between 6-12Mohms with a Narishige MF-9 microforge (Narishige International USA,East Meadow, N.Y.). The pipettes were filled with an intracellularsolution containing (in mM): 135 KCl, 10 NaCl, 5 EGTA, 10 Hepes, pH wasadjusted to 7.4 with NMDG (N-Methyl-D-Glucamine).

The cultured Calu-3 cells were placed in a chamber containing thefollowing extracellular solution (in mM): 135 NaCl, 5 KCl, 1.2 CaCl2,0.5 MgCl2 and 10 Hepes (free acid), pH was adjusted to 7.4 with NMDG.

Cells were viewed using the 40×DIC objective of an Olympus IX71microscope (Olympus Inc., Tokyo, Japan). After a cell-attached gigasealwas established, a gentle suction was applied to break in, and to attainthe whole-cell configuration. Immediately upon breaking in, the cell wasvoltage clamped at −120, −60, −40 and 0 mV, and was stimulated withvoltage steps between ±100 mV (500 ms/step). After collecting thewhole-cell currents at the control condition, the same cell was perfusedthrough bath with the test fluid comprising same exatracelluar solutesand pH as for the above control fluid, and whole-cell currents atdifferent holding potentials were recorded with the same protocols.

Electrophysiological data were acquired with an Axon Patch 200Bamplifier, low-pass filtered at 10 kHz, and digitized with 1400ADigidata (Axon Instruments, Union City, Calif.). The pCLAMP 10.0software (Axon Instruments) was used to acquire and to analyze the data.Current (I)-to-voltage (V) relationships (whole cell conductance) wereobtained by plotting the actual current value at approximately 400 msecinto the step, versus the holding potential (V). The slope of the I/Vrelationship is the whole cell conductance.

Drugs and Chemicals.

Whenever indicated, cells were stimulated with a cAMP stimulatorycocktail containing 8-Br-cAMP (500 mM), IBMX (isobutyl-1-methylxanthie,200 mM) and forskolin (10 mM). The cAMP analog 8-Br-cAMP (Sigma Chem.Co.) was used from a 25 mM stock in H2O solution. Forskolin (Sigma) andIBMX (Sigma) were used from a DMSO solution containing both 10 mMForskolin and 200 mM IBMX stock solution.

Patch Clamp Results:

FIG. 100 shows whole-cell currents under basal (no cAMP) conditions,with a protocol stepping from zero mV holding potential to +/−100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance, obtainedfrom the current-to-voltage relationships is highly linear under bothconditions, and reflects a modest, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug(inventive electrokinetically generated fluid) is also linear, and thereversal potential is near zero mV. There is a decrease in the wholecell conductance under hyperpolarizing conditions.

FIG. 101 shows whole-cell currents under basal conditions, with aprotocol stepping from −40 mV holding potential to ±100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance obtained fromthe current-to-voltage relationships is highly linear under bothconditions, and reflects a modest, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug(inventive electrokinetically generated fluid) is also linear, and thereversal potential is near zero mV. Values are comparatively similar tothose obtained with the zero mV protocol.

FIG. 102 shows whole-cell currents under basal conditions, with aprotocol stepping from −60 mV holding potential to ±100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance obtained fromthe current-to-voltage relationships is highly linear under bothconditions, and reflects a minor, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug isalso linear, and the reversal potential is near zero mV. Values arecomparatively similar to those obtained with the zero mV protocol.

FIG. 103 shows whole-cell currents under basal conditions, with aprotocol stepping from −120 mV holding potential to ±100 mV.Representative tracings are the average of n=12 cells. The tracings onthe left are the control, followed by the whole-cell tracings whileperfusing the test solution (middle). The tracings on the right are thecomposite delta obtained by subtraction of the test average values, fromthose under control conditions. The whole-cell conductance obtained fromthe current-to-voltage relationships is highly linear under bothconditions, and reflects a minor, albeit significant change inconductance due to the test conditions. The contribution to thewhole-cell conductance, i.e., the component inhibited by the drug isalso linear, and the reversal potential is near zero mV. Values arecomparatively similar to those obtained with the zero mV protocol.

FIG. 104 shows whole-cell currents under cAMP-stimulated conditions,obtained with protocols stepping from various holding potentials to ±100mV. Representative tracings are the average of n=5 cells. The tracingson the left are the control, followed by the whole-cell tracings aftercAMP stimulation, followed by perfusion with the drug-containingsolution. The tracings on the right are the composite delta obtained bysubtraction of the test average values in drug+cAMP, from those undercontrol conditions (cAMP alone). The tracings on the Top are thoseobtained from voltage protocol at zero mV, and the ones below, at −40mV. The whole-cell conductance obtained from the current-to-voltagerelationships is highly linear under all conditions, and reflects achange in conductance due to the test conditions.

FIG. 105 shows whole-cell currents under cAMP-stimulated conditions,obtained with protocols stepping from various holding potentials to ±100mV. Representative tracings are the average of n=5 cells. The tracingson the left are the control, followed by the whole-cell tracings aftercAMP stimulation, followed by perfusion with the drug-containingsolution. The tracings on the right are the composite delta obtained bysubtraction of the test average values in drug+cAMP, from those undercontrol conditions (cAMP alone). The tracings on the Top are thoseobtained from voltage protocol at −60 mV, and the ones below, at −120mV. The whole-cell conductance, obtained from the current-to-voltagerelationships, is highly linear under all conditions, and reflects achange in conductance due to the test conditions.

FIG. 106 shows the effect of holding potential on cAMP-activatedcurrents. The effect of the drug (the inventive electrokineticallygenerated fluids; RNS-60; electrokinetically treated normal salinecomprising 60 ppm dissolved oxygen) on the whole-cell conductance wasobserved under different voltage protocols (0, −40, −60, −120 mV holdingpotentials). Under basal conditions, the drug-sensitive whole-cellcurrent was identical at all holding potentials (voltage-insensitivecontribution, Top Left panel). In the cAMP-activated conditions,however, the drug-sensitive currents were much higher, and sensitive tothe applied voltage protocol. The current-to-voltage relationships arehighly nonlinear. This is further observed in the subtracted currents(Bottom panel), where the contribution of the whole cell conductance atzero mV was further subtracted for each protocol (n=5).

Summary of Example.

According to particular aspects, therefore, the data indicate that thereis a modest but consistent effect of the drug (the inventiveelectrokinetically generated fluids; RNS-60; electrokinetically treatednormal saline comprising 60 ppm dissolved oxygen) under basalconditions. To enhance the effect of the drug on the whole-cellconductance, experiments were also conducted by perfusing the drug afterstimulation with a cAMP stimulating “cocktail”, which dramaticallyincreased the whole-cell conductance. Interestingly, this protocol alsoincreased the drug-sensitive portion of the whole-cell conductance,which was ten-times higher than that observed under basal conditions.Additionally, in the presence of cAMP stimulation, the drug showeddifferent effects with respect to the various voltage protocols,indicating that the electrokinetically generated fluids affect avoltage-dependent contribution of the whole-cell conductance. There wasalso a decrease in a linear component of the conductance, furthersuggesting at least a contribution of the drug to the inhibition ofanother pathway (e.g., ion channel, voltage gated cation channels,etc.).

In particular aspects, and without being bound by mechanism, Applicants'data are consistent with the inventive electrokinetically generatedfluids (e.g., RNS-60; electrokinetically treated normal salinecomprising 60 ppm dissolved oxygen) producing a change either on achannel(s), being blocked or retrieved from the plasma membrane.

Example 24 Patch Clamp Analysis Conducted on Calu-3 Cells Perfused withInventive Electrokinetically Generated Fluids (RNS-60 and Solas)Revealed that (i) Exposure to RNS-60 and Solas Resulted in Increases inWhole Cell Conductance, (ii) that Exposure of Cells to the RNS-60Produced an Increase in a Non-Linear Conductance, Evident at 15 minIncubation Times, and (iii) that Exposure of Cells to the RNS-60Produced an Effect of RNS-60 Saline on Calcium Permeable Channels

Overview.

In this Example, patch clamp studies were performed to further confirmthe utilities, as described herein, of the inventive electrokineticallygenerated saline fluids (RNS-60 and Solas), including the utility tomodulate whole-cell currents. Two sets of experiments were conducted.

The summary of the data of the first set of experiments indicates thatthe whole cell conductance (current-to-voltage relationship) obtainedwith Solas saline is highly linear for both incubation times (15 min, 2hours), and for all voltage protocols. It is however evident, thatlonger incubation (2 hours) with Solas increased the whole cellconductance. Exposure of cells to the RNS-60 produced an increase in anon-linear conductance, as shown in the delta currents (Rev-Solsubtraction), which is only evident at 15 min incubation time. Theeffect of the RNS-60 on this non-linear current disappears, and isinstead highly linear at the two-hour incubation time. The contributionof the non-linear whole cell conductance, as previously observed, wasvoltage sensitive, although present at all voltage protocols.

The summary of data of the second set of experiments indicates thatthere is an effect of the RNS-60 saline on a non-linear current, whichwas made evident in high calcium in the external solution. Thecontribution of the non-linear whole cell conductance, although voltagesensitive, was present in both voltage protocols, and indicates aneffect of RNS-60 saline on calcium permeable channels.

First Set of Experiments Increase of Conductance; and Activation of aNon-Linear Voltage Regulated Conductance

Methods for First Set of Experiments:

See EXAMPLE 23 for general patch clamp methods. In the following firstset of experiments, patch clamp studies were performed to furtherconfirm the utility of the inventive electrokinetically generated salinefluids (RNS-60 and Solas) to modulate whole-cell currents, using Calu-3cells under basal conditions, with protocols stepping from either zeromV holding potential, −120 mV, or −60 mV.

The whole-cell conductance in each case was obtained from thecurrent-to-voltage relationships obtained from cells incubated foreither 15 min or two hours, to further confirm the results of EXAMPLE23. In this study, groups were obtained at a given time, for eitherSolas or RNS-60 saline solutions. The data obtained are expressed as themean±SEM whole cell current for 5-9 cells.

Results:

FIGS. 117A-C show the results of a series of patch clamping experimentsthat assessed the effects of the electrokinetically generated fluid(e.g., RNS-60 and Solas) on epithelial cell membrane polarity and ionchannel activity at two time-points (15 min (left panels) and 2 hours(right panels)) and at different voltage protocols (A, stepping fromzero mV; B, stepping from −60 mV; and C, stepping from −120 mV). Theresults indicate that the RNS-60 (filled circles) has a larger effect onwhole-cell conductance than Solas (open circles). In the experimentsimilar results were seen in the three voltage protocols and at both the15 minute and two-hour incubation time points.

FIGS. 118A-C show graphs resulting from the subtraction of the Solascurrent data from the RNS-60 current data at three voltage protocols(“Delta currents”) (A, stepping from zero mV; B, stepping from −60 mV;and C, stepping from −120 mV) and the two time-points (15 mins (opencircles) and 2 hours (filled circles)). These data indicated that at the15 minute time-point with RNS-60, there is a non-linearvoltage-dependent component that is absent at the 2 hour time point.

As in previous experiments, data with “Normal” saline gave a veryconsistent and time-independent conductance used as a reference. Thepresent results were obtained by matching groups with either Solas orRNS-60 saline, and indicate that exposure of Calu-3 cells to the RNS-60saline under basal conditions (without cAMP, or any other stimulation),produces time-dependent effect(s), consistent with the activation of avoltage-regulated conductance at shorter incubation times (15 min). Thisphenomenon was not as apparent at the two-hour incubation point. Asdescribed elsewhere herein, the linear component is more evident whenthe conductance is increased by stimulation with the cAMP “cocktail”.Nonetheless, the two-hour incubation time showed higher linearconductance for both the RNS-60 and the Solas saline, and in this case,the RNS-60 saline doubled the whole cell conductance as compared toSolas alone. This evidence indicates that at least two contributions tothe whole cell conductance are affected by the RNS-60 saline, namely theactivation of a non-linear voltage regulated conductance, and a linearconductance, which is more evident at longer incubation times.

Second Set of Experiments Effect on Calcium Permeable Channels

Methods for Second Set of Experiments:

See EXAMPLE 23 for general patch clamp methods. In the following secondset of experiments, yet additional patch clamp studies were performed tofurther confirm the utility of the inventive electrokineticallygenerated saline fluids (RNS-60 and Solas) to modulate whole-cellcurrents, using Calu-3 cells under basal conditions, with protocolsstepping from either zero mV or −120 mV holding potentials.

The whole-cell conductance in each case was obtained from thecurrent-to-voltage relationships obtained from cells incubated for 15min with either saline. To determine whether there is a contribution ofcalcium permeable channels to the whole cell conductance, and whetherthis part of the whole cell conductance is affected by incubation withRNS-60 saline, cells were patched in normal saline after the incubationperiod (entails a high NaCl external solution, while the internalsolution contains high KCl). The external saline was then replaced witha solution where NaCl was replaced by CsCl to determine whether there isa change in conductance by replacing the main external cation. Underthese conditions, the same cell was then exposed to increasingconcentrations of calcium, such that a calcium entry step is made moreevident.

Results:

FIGS. 119A-D show the results of a series of patch clamping experimentsthat assessed the effects of the electrokinetically generated fluid(e.g., Solas (panels A and B) and RNS-60 (panels C and D)) on epithelialcell membrane polarity and ion channel activity using different externalsalt solutions and at different voltage protocols (panels A and C showstepping from zero mV, whereas panels B and D show stepping from −120mV). In these experiments one time-point of 15 minutes was used. ForSolas (panels A and B) the results indicate that: 1) using CsCl (squaresymbols) instead of NaCl as the external solution, increased whole cellconductance with a linear behavior when compared to the control (diamondsymbols); and 2) CaCl₂ at both 20 mM CaCl₂ (circle symbols) and 40 mMCaCl₂ (triangle symbols) increased whole cell conductance in anon-linear manner. For RNS-60 (panels C and D), the results indicatethat: 1) using CsCl (square symbols) instead of NaCl as the externalsolution had little effect on whole cell conductance when compared tothe control (diamond symbols); and 2) CaCl₂ at 40 mM (triangle symbols)increased whole cell conductance in a non-linear manner.

FIGS. 120A-D show the graphs resulting from the subtraction of the CsClcurrent data (shown in FIG. 119) from the 20 mM CaCl₂ (diamond symbols)and 40 mM CaCl₂ (square symbols) current data at two voltage protocols(panels A and C, stepping from zero mV; and B and D, stepping from −120mV) for Solas (panels A and B) and RNS-60 (panels C and D). The resultsindicate that both Solas and RNS-60 solutions activated acalcium-induced non-linear whole cell conductance. The effect wasgreater with RNS-60 (indicating a dosage responsiveness), and withRNS-60 was only increased at higher calcium concentrations. Moreover,The non-linear calcium dependent conductance at higher calciumconcentration was also increased by the voltage protocol.

The data of this second set of experiments further indicates an effectof RNS-60 saline and Solas saline for whole cell conductance dataobtained in Calu-3 cells. The data indicate that 15-min incubation witheither saline produces a distinct effect on the whole cell conductance,which is most evident with RNS-60, and when external calcium isincreased, and further indicates that the RNS-60 saline increases acalcium-dependent non-linear component of the whole cell conductance.

The accumulated evidence suggests activation by Revalesio saline of ionchannels, which make different contributions to the basal cellconductance.

Taken together with Applicants' other data (e.g., the data of Applicantsother working Examples) particular aspects of the present inventionprovide compositions and methods for modulating intracellular signaltransduction, including modulation of at least one of membranestructure, membrane potential or membrane conductivity, membraneproteins or receptors, ion channels, lipid components, or intracellularcomponents with are exchangeable by the cell (e.g., signaling pathways,such as calcium dependant cellular signaling systems, comprising use ofthe inventive electrokinetically generated solutions to impartelectrochemical and/or conformational changes in membranous structures(e.g., membrane and/or membrane proteins, receptors or other membranecomponents) including but not limited to GPCRs and/or g-proteins.According to additional aspects, these effects modulate gene expression,and may persist, dependant, for example, on the half lives of theindividual messaging components, etc.

Example 25 Atomic Force Microscopy (AFM) Measurements of the InventiveElectrokinetic Fluid (RNS-60) Indicated the Presence and/or Formation ofHydrophobic Surface Nanobubbles that were Substantially Smaller thatThose Present in Control ‘Pressure Pot’ (PNS-60) Fluid

Overview.

Applicants used Atomic Force Microscopy (AFM) measurements tocharacterize hydrophobic nanobubbles in the inventive electrokineticfluid (RNS-60).

Materials and Methods:

AFM Studies.

AFM studies were preformed at an art-recognized Nanotech User Facility(NTUF). For AFM studies, a very small and sensitive needle is dippedinto a droplet of water placed onto a hydrophobic surface. The needlethen scans over the water/surface interface at rates such as 1 mm² in˜15 minutes. The needle records any imperfections in the surfacegeometry, and is sensitive enough to record the presence of smallbubbles.

The Silicon substrate upon which the water droplets were placed wasprepared using Trichloro(1H,1H,2H,2H-perfluorooctyl)silane), and theresulting hydrophobic surface causes water to bead up with contactangles of approximately 95 degrees. This coating is used in many AFMstudies, in part, because it is particularly durable.

Solution Preparation.

Two test solutions were studied: RNS-60 and PNS-60. RNS-60 is aninventive electrokinetic fluid comprising 60 ppm oxygen, whereas PNS-60is a non-electrokinetic control fluid comprising 60 ppm oxygen preparedby conventional exposure to a pressurized oxygen head (i.e., pressurepot oxygenated fluid). Each test solution was initially buffered byaddition of a small amount of neutral phosphate buffer (pH 7) solution,and approximately 60-70 uL of each buffered test solution (approximately22° C.) was placed onto a previously prepared silica plate.

Results:

Under AFM, the RNS-60 droplet displayed a distribution of about 20hydrophobid nanobubbles in a 1 mm² area, having dimensions of ˜20 nmwide and 1.5 nm tall or smaller (FIG. 121A). By contrast, under AFM, thePNS-60 droplet displayed approx 5 hydrophobic nanobubbles in a 1 mm²area, having dimensions of ˜60 nm wide and ˜5 nm tall (FIG. 121B). ThePNS-60 droplet, therefore, had much fewer and much larger hydrophobicnanobubbles compared to the RNS60 droplet.

According to particular aspects, therefore, there is a substantialdifference in the size and distribution of hydrophobic surfacenanobubbles between the RNS-60 and PNS-60 test solutions, where thenanobubbles are either initially present in, and/or formed within thetest fluids during AFM measurement.

As discussed elsewhere herein, according to particular aspects of thepresent invention, the inventive electrokinetically altered fluidscomprise an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide, upon contact ofa living cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity.

Applicants point out, however, that the hydrophobic bubbles (forming ona hydrophobic surface), such as those observed in AFM experiments arelikely fundamentally different from inventive biologically-activecharge-stabilized nanostructure disclosed herein. According toparticular aspects therefore, while the AFM experiments in this workingExample support, based on the size and distribution hydrophobic bubbleformation, that the inventive electrokinetic fluids (e.g., RNS-60) arefundamentally distinct from non-electrokinetic control fluids, thehydrophobic bubbles are likely distinct from and/or derived from theinventive charge-stablilized oxygen-containing nanostrutures describedin detail elsewhere herein. In any event, relative to the inventiveelectrokinetic fluids, control pressure pot oxygenated fluids do notcomprise charge-stabilized oxygen-containing nanostructures capable ofmodulation of at least one of cellular membrane potential and cellularmembrane conductivity.

Example 26 Analysis of Inventive Fluid on Growth Inhibition ofPseudomonas on Plates

Applying gas-enriched saline solution of the present invention limitsPseudomonas growth. These tests were performed using an inventiveoxygen-enriched saline solution.

Two test strains of Pseudomonas (ATCC accession no. 10145 and ATCCaccession no. 27853) were prepared from fresh 24-hour cultures to aMcFarland 1 concentration (approximately 3×10⁸ microorganisms/mL). Eachof the bacterial aliquots (1 mL) was serially diluted in 10 folddilutions in 9 mL of broth-saline made from 1 part TSB broth and 9 partssterile saline. The bacterial concentrations tested were 10⁷, 10⁶, 10⁵,10⁴, 10³, and 10². A negative control tube (no bacteria and nogas-enriched fluid of the present invention) was prepared. The positivecontrol tubes (containing no gas-enriched fluid, normal saline and eachof the 6 bacterial concentrations) were included in each set of tubesfor testing of each bacterial strain.

Using the 10 fold dilutions of each bacterial strain, a set of 36 tubeswas inoculated as follows:

-   -   Six tubes received 1 mL of each of the dilutions; then 4 mL of        each of the test gas-enriched fluids were added to each tube        (bringing the total volume to 5 mL). The gas-enriched fluids        were labeled as 50 ppm, 40 ppm, 30 ppm, 20 ppm, 10 ppm, and        normal saline. The positive control was normal saline, against        which all of the other tubes were measured for growth.

All tubes of each 36-tube experiment set for each Pseudomonas specieswere measured serially at 2 hour intervals within a 24 hour period afterinitial incubation at 35° C. Between readings of each set, the tubeswere returned to 35° C. for continued incubation. Readings wereperformed using a calibrated spectrophotometer set at OD₅₄₀.

The results of these tests indicate that the gas-enriched fluid of thepresent invention positively inhibits Pseudomonas strains at severalbacterial dilutions within 4 to 12 hours after the start of incubationat 35° C., as well as later (16-24 hours) for both strains tested.

Specifically, the highest positive inhibition was found during 16 to 24hours for both strains, at 30 or 50 concentration. The positiveinhibition varies slightly depending on the concentration of testsolutions, and concentration of bacterial samples tested.

TABLE 10 Time at positive inhibition of Pseudomonas Time (hours) 2 4 6 810 12 16 18 20 22 24 ATCC 27853 0 1 3 1 2 2 5 0 5 7 6 % + — 3 10 3 6 616 — 16 23 20 ATCC 10145 0 4 3 4 3 3 2 1 5 6 8 % + — 13 10 13 10 10 6 316 20 26 % + = number +/30 (6 bacterial concentrations × 5 solutionconcentrations)

Example 27 MIC Studies on Pseudomonas Growth Inhibition in Tubes

Minimum inhibitory concentration (MIC) test solutions in two-folddilutions were prepared in a base of broth-saline mixture made from 1part TSB broth and 4 parts CFU-NS. The negative control tube (containingno bacteria and no gas-enriched fluid) and the positive control tube(containing no gas-enriched fluid and bacteria) were included in eachset of tubes tested. The fluid dilutions were: 50, 25, 12.5, 6.25, 3.12,1.55, and 0.7 ppm, respectively. Following preparation of the tubes foreach bacterial strain, pH was measured on samples from the twosolutions. CFU-gas-enriched fluid pH was about 6.8-7.2, while CFU-NS waspH about 6.2.

Following preparation of the 2 sets of covered tubes at 35° C. for 18hours, visual inspection of all tubes revealed that the first tubelabeled 50 in each set showed no growth and all other tubes (except thenegative control tube) showed moderate growth.

The tubes labeled 50 and showed no growth upon visual inspection fromthe MIC studies were further tested for minimum bactericidalconcentrations (MBC). Samples were taken from each of these two tubesusing sterile calibrated disposable loops of 0.1 mL capacity and wereimmediately streaked on BA plates to detect growth or inhibition ofgrowth on the challenge bacteria. Following 18 hours incubation at 35°C., samples collected from each of the tubes showing no visual growth(tube 50 from ATCC 10145 and tube 50 from ATCC 27853) both showed growthat very high levels when streaked and plated on BA plates. The numbersof colonies were too numerous to count. Thus, a slight delay in growthwas experienced with the tube 50 gas-enriched fluids.

Example 28 Analysis of Inventive Fluid on Growth Inhibition ofPseudomonas in Dressings

Aquacel dressings were tested dry and hydrated with either test fluids52 (pH 7.2-7.8), 50 (pH 6.0-6.2), 42 (pH 7.2), 34 (pH 7.2), 25 (pH 6.8),and 10 (pH 6.2), or normal saline (pH 6.2), against Pseudomonas strainsATCC accession no. 10145 and ATCC accession no. 27853 A firstapplication of the gas-enriched test fluids or normal saline fluid tothe dressings (0.4 mL) was followed by a second application 12 hourslater (0.25 mL).

Results revealed a 3-4 mm clear area of inhibition around one of thethree dressing pieces (1 cm square) treated with sterile 25 fluidapplied to the three Pseudomonas ATCC accession no. 10145 seeded plates,and a 3-4 mm clear area of inhibition around one of the three dressingpieces (1 cm square) treated with sterile 25 fluid applied to one of thethree Pseudomonas ATCC accession no. 27853 seeded plates. A clear areaof less than 1-2 mm of inhibition was around 2 sides of the dry dressingon both strains. No zones of inhibition were detected around the othertest or control dressings.

Example 29 Analysis of Inventive Fluid on Growth Inhibition ofMicroorganisms in Dressings

Blood Agar plates were used and wound dressings used were PromogramPrisma matrix dressing (A) and hydrofiber dressing (B). The steriledressings were tested with microorganisms from a 24 hour culture at acell density with McFarland equivalence turbidity standards of3.0×10⁸/mL.

Test microorganisms were Staphlycoccus aureaus, Staphylococcusepidermidis, Pseudomonas aeruginosa, E. coli, and Candida albicans.

Dressings of each type were hydrated with approximately 0.8 mL of eithergas-enriched fluid or normal saline, and left for approximately 30minutes. Dry dressings were used as controls. Plates were then grown for24 hours and results recorded.

As viewed in the cell plates, the hydrofiber dressing (B) had no effecton growth of test microorganisms whether dry or hydrated with eitherfluid. The Prisma dressing (A) hydrated with gas-enriched fluid showed azone of partial inhibition with colonies for Staph. aureaus, a 1 mm zoneof inhibition for Staph. epidermidis, a 2-3 mm zone of inhibition withan added halo effect extending out an additional 2-3 mm for Pseudomonas,a 2 mm zone of inhibition for Candida, and no effect for E. coli.

The dry control hydrofiber dressing (A) had a zone of 2-3 mm ofinhibition for Staph. auereaus, Staph. epidermidis, Pseudomonas, and apartial inhibition of about 1-2 mm for E. coli and Candida, withbreak-through colonies observed.

The normal saline control hydrofiber dressing (A) showed a 2 mm zone ofpartial inhibition for Staph. auereaus, Staph. epidermidis, andPseudomonas. Colonies were observed grown into E. coli and Candida teststrains.

Example 30 MIC/MBC Testing with Tobramycin

The gas-enriched fluids of the present invention were tested randomlyagainst three Pseudomonas strains, PA 01, PA 14, and PA K. Eachbacterial culture was prepared from fresh 24-hour cultures to McFarland1 concentration (approximately 3×10⁸ microorganisms/mL). Each of thebacterial samples (1 mL) were then serially diluted in 10 fold dilutionsto 9 mL of a broth-saline mixture made from 1 part MH broth and 9 partssterile saline. The bacterial concentrations tested were 10⁵, 10⁴, 10³,and 10². Negative control tubes (containing no bacteria and nogas-enriched saline solution, and no Tobramycin), as well as positivecontrol tubes (containing test bacteria concentrations and normal salinebut no Tobramycin, and no gas-enriched fluid), were run side-by-sidewith the test solutions. Cells were incubated at 35° C. Results areshown in Table 11 below. Sheared normal saline was passed through thegas-enriching diffuser device of the present invention without theaddition of a gas to the fluid.

TABLE 11 Better kill rate with Tobramycin and Gas-enriched fluid thanTobramycin Alone Dissolved Gas Fluid Description (Oxygen, ppm) PA01 PA14PAK A Gas-enriched Saline 33.4 Yes Yes No B Gas-enriched saline 23.8 NoYes No C Gas-enriched saline 49.6 Yes Yes Yes D Normal saline 9.6 No NoNo E Gas-enriched saline 42.8 Yes Yes Yes F Sheared normal saline (no14.1 Yes Yes Yes gas enrichment)

Test results shown in FIG. 39 illustrate the results of testingPseudomonas growth inhibition with deionized water, an oxygen-enrichedfluid of the present invention, as well as normal saline.

Example 31 Analysis of Corneal Fibroblast Proliferation

Sterile gas-enriched water (with oxygen content of approximately 50ppm), or standard sterile deionized water was utilized in preparing cellculture media. A human corneal stromal fibroblast cell line was platedin 24 well tissue culture plates at a density of 1×10⁵ cells/cm² andcells were tested for viability using a standard Live/Dead assay fromMolecular Probes, Inc. Viability tests were conducted at 1 or 3 days ofcontinuous culture with media changes twice per day. Viable andnon-viable cells were counted in 10 random 20× fields (0.2 mm²area/field) in each well. The percentage of dead cells was thencalculated. Cells in the inventive gas-enriched media had fewer deadcells than in standard culture media. Results are shown in FIG. 46.

Example 32 Assessment of Gas-Enriched Fluid on KeratoconjunctivitisSicca

According to particular aspects of the present invention, upon theinitial visitation, subjects are be examined, and systemic andophthalmic histories reviewed, including current use of concomitantmedications and/or therapies. Slit lamp examination is conducted by wayof assessment of corneal and conjunctival staining, assessment of tearvolume, and assessment of bulbar conjunctival erythema. Tearscopeexamination is conducted by assessment of inferior tear meniscus height,and stability and thickness of tear film. Treatment is dispensed basedon randomization into the following groups:

a. Treatment group: Gas-Enriched fluid instilled four times per day for6 weeks.

b. Control group: non-preserved saline instilled four times per day for6 weeks.

Upon the second visitation following a 2 week interval (+/−2 days), eachsubject completes a dry eye questionnaire, and the subject's systemicand ophthalmic history, as well as any change in medications and/ortherapies will be reviewed. Slit lamp examination is conducted byassessment of corneal and conjunctival staining, assessment of tearvolume, and assessment of bulbar conjunctival erythema. Tearscopeexamination is conducted by assessment of inferior tear meniscus heightas well as stability and thickness of tear film.

Upon the third examination following a 4 week interval (+/−2 days), eachsubject completes a dry eye questionnaire, and the subject's system andophthalmic history is reviewed, as well as any change in medicationsand/or therapies. Slit lamp examination is conducted by assessment ofcorneal and conjunctival staining, assessment of tear volume, andassessment of bulbar conjunctival erythema. Tearscope examination isconducted by assessment of inferior tear meniscus height and stabilityand thickness of tear film.

Upon the fourth examination following a 6 week interval (+/−2 days),each subject completes a dry eye questionnaire, and the subject's systemand ophthalmic history is reviewed, as well as any change in medicationsand/or therapies. Slit lamp examination is conducted by assessment ofcorneal and conjunctival staining, assessment of tear volume, andassessment of bulbar conjunctival erythema. Tearscope examination isconducted by assessment of inferior tear meniscus height and stabilityand thickness of tear film. In addition, a treatment cross-over isevaluated according to previous treatment solutions collected, newtreatment dispensed, and patients counseled to begin using new therapyimmediately.

Upon the fifth examination following a 2 week interval (+/−2 days), eachsubject completes a dry eye questionnaire, and the subject's system andophthalmic history is reviewed, as well as any change in medicationsand/or therapies. Slit lamp examination is conducted by assessment ofcorneal and conjunctival staining, assessment of tear volume, andassessment of bulbar conjunctival erythema. Tearscope examination isconducted by assessment of inferior tear meniscus height and stabilityand thickness of tear film.

Upon the sixth examination following a 4 week interval (+/−2 days), eachsubject completes a dry eye questionnaire, and the subject's system andophthalmic history is reviewed, as well as any change in medicationsand/or therapies. Slit lamp examination is conducted by assessment ofcorneal and conjunctival staining, assessment of tear volume, andassessment of bulbar conjunctival erythema. Tearscope examination isconducted by assessment of inferior tear meniscus height and stabilityand thickness of tear film.

Upon the seventh examination following a 6 week interval (+/−2 days),each subject completes a dry eye questionnaire, and the subject's systemand ophthalmic history is reviewed, as well as any change in medicationsand/or therapies. Slit lamp examination is conducted by assessment ofcorneal and conjunctival staining, assessment of tear volume, andassessment of bulbar conjunctival erythema. Tearscope examination isconducted by assessment of inferior tear meniscus height and stabilityand thickness of tear film, and subjects are exited from the study.

No intitiation or occasional use of new topically applied medication orsystemic medication is allowed by subjects, specifically including butnot limited to antihistamines, aspirin-related drugs, steroids,Paquenil™ or psychoactive/anti-depressant medication. Subjects currentlyusing this medication on a continual basis are allowed. Additionally,subjects with a history of glaucoma, anterior membrane dystrophy, activetrichiasis or any eyelid globe malposition abnormality, e.g. entropion,ectropion, etc. are allowed to participate. Keratoconjunctivitis siccasecondary to the destruction of conjunctival goblet cells as withvitamin A deficiency or scarring such as that with cicatricialpemphigoid, libal stem cell deficiency, graft vs. host disease, alkaliburns, Stevens-Johnson syndrome, trachoma, or irradiation are allowed toparticipate. Finally, uncontrolled systemic disease or the presence ofany significant illness or condition that could, in the judgment of theinvestigator, interfere with interpretation of the study results (suchas Sjogren's syndrome and related autoimmune diseases are allowed) areallowed. In the event that a subject becomes pregnant, the studyimmediately terminates for that subject.

Example 33 Adjunct Therapy for Keratoconjunctivitis Sicca

According to additional aspects of the present invention, the treatmentdispensed in this study is used as an adjunct to all habitual topicaland oral medications being used. All patients enrolled continue takingmedications as prescribed previously.

Following recruitment, test subjects are randomized to either theGas-enriched saline treatment group or the non-preserved saline(control) group. Subjects are masked as to the treatment therapy theyare receiving (treatment gas-enriched or control). The clinical observerperforming the objective measurements is masked to the treatment groupas well.

Sample size calculations and statistical methods to detect significantchanges in the outcome measures are derived from previous comparableclinical trials. In order to detect a significant change compared tocontrol with a power of 0.8, a minimum of 25 subjects is required. Forall datasets, normality and equal variance assumption testing isperformed using the Kolmogorov-Smirnov Test and the Levene median tests.The results of all outcome measures are compared, significance (p<0.05)for each treatment group compared to baseline values with one-wayrepeated measure analysis of variance and subsequently with a post hocmultiple comparison test (Student-Newman-Keuls test) using Sigma StatSoftware (Systat Software, Inc., San Jose, Calif.). In addition, thetest and control groups are compared with two-way repeated measureanalysis of variance.

Example 34 Clinical Assessments of Dry Eyes

According to additional aspects of the present invention, clinicalscreening procedures are effectuated, based on health history of botheyes by observing redness, pain, irritation, visual change, andcompliance. As a subjective assessment, each subject are asked tocomplete the Ocular Surface Disease Index Dry Eye Questionnaire at eachvisit. The OSDI uses 12 questions to address the subject's symptoms andvisual impact. For each question, the index utilizes a grading scale of0-4 as follows: 0 if none of the time; 1 if some of the time; 2 if halfof the time; 3 if most of the time; 4 if all of the time.

As a subjective assessment, each subject is asked to complete a SnellenVA measurement at 20 feet, as well as undergo a slit lamp examination toassess corneal staining with sodium fluorescein, conjunctival stainingwith lissamine green and bulbar conjunctival erythema. All grading isusing standard grading scales of 0-4 as described below. In addition,tearscope examination using Keeler Tearscope is used to measure theheight of the inferior tear meniscus, tear break up time and thicknessof tear film. In the absence of any historical or clinical(subjective/objective) abnormalities, participation in the study iscontinued.

TABLE 12 Brief Noninvasive Clinical Assessment Schedule Study for Visits1-7 1. Vision Snellen distance acuity at 20 feet, Room #6, B1, AstonCenter 2. Questionnaire Ocular Surface Disease Index 3. Ocular SurfaceCorneal staining (0-4) Bulbar conjunctival staining (0-4) Bulbarconjunctival erythema (0-4) 4. Tear Film Height of inferior tearmeniscus* Tear break up time* Tear film thickness* Corneal staining: Theextent of corneal staining by sodium fluorescein is graded according tothe scale below for each quadrant: 1 central, 2 superior, 3 temporal, 4nasal, 5 inferior. 0: No staining 1: Trace 2: Mild 3: Moderate 4: SevereConjunctival staining: The extent of bulbar conjunctival staining bylissamine green is graded according to the scale below for eachquadrant: 0: No staining 1: Trace 2: Mild 3: Moderate 4: Severe

Erythema: The extent of conjunctival erythema is assessed according tothe scale below: 0: No redness 1: Trace 2: Mild 3: Moderate 4: Severe

*Height of inferior tear meniscus, tear break up time and tear filmthickness is assessed using a Tearscope.

Tearscope:

A Keeler Tearscope (Keeler, Broomall, Pa.) is used to analyze tearinterference patterns using white light. For image acquisition, adigital camera, or other image capturing device, is coupled to acomputer will be used to record changes in tear interference patterns.The digitally recorded images are analyzed to assess the lipid tearpattern, the stability of the tear film, and the tear film thickness.

Stability of the tear film is assessed non-invasively by measurements oftear break up time. For tear film thickness, a computer generated (ADOBEPhotoshop, ADOBE Systems, San Jose, Calif.) thickness chart using amap-color intensity scale is used to covert light reflectance into nm.

Example 35 Therapy for Allergy Conjunctiva

Allergen challenge will be done using our standard protocol to confirmthe allergic subject's level of reactivity according to Table 13.

TABLE 13 Allergen challenge protocol Relative Quantitative AllergenConcentration Concentration Cat Dander Low 2500 AU/ml Medium 5000 AU/mlHigh 10,000 AU/ml Dust Mite Low 2500 AU/ml Medium 5000 AU/ml High 10,000AU/ml Grass Pollen Low 10,000 PNU/ml Medium 20,000 PNU/ml High 40,000PNU/ml Ragweed Pollen Low 10,000 PNU/ml Medium 20,000 PNU/ml High 40,000PNU/ml

According to the concentration chart, the investigator determines theallergen threshold dose starting with the lowest dose of allergen andtitrating to the medium and then highest dose at 15-minute intervalsuntil a positive response is achieved as follows:

1. one drop of allergen in the required concentration is instilled intothe conjunctival sac of each eye.

2. At 5, 10, 15, and 20 minutes following challenge, the eye symptomassessments (itching and redness only) is performed.

3. For the symptom of itching in each eye, if a score of greater than orequal to 2+ on the severity scale is obtained using the Lowconcentration of allergen on at least one of the time points (5, 10, 15,and 20 minutes), this is considered a positive response. Both eyes donot need to obtain a positive response at the same time point.

4. For the symptom of redness in each eye, if a score of greater than orequal to 2+ on the severity scale is obtained using the Lowconcentration of allergen on at least one of the time points (5, 10, 15,and 20 minutes), this is considered a positive response. Both eyes donot need to obtain a positive response at the same time point.

5. If a negative response is obtained using the Low concentration ofallergen, i.e. less than 2+ on the itching and redness severity scale,then the challenge is repeated as above at the medium, and if necessary,at the high concentration of the allergen. Once a positive result isobtained, then this concentration is recorded as the appropriateallergen dose, and used at all subsequent visits.

Standardized extracts of cat dander, dust mite, grass pollen or ragweedpollen is used. Three different concentrations of allergen may be usedin order to establish the lowest concentration which elicits a moderateconjunctival allergic response. Conjunctival erythema, edema, anditching is graded on a scale of 0 to 4 according to the following scale:

Edema:

-   0=None.-   1=Mild: slight lower or upper lid swelling.-   2=Moderate: noticeable swelling of lower and/or upper lids.-   3=Severe: visible swelling; difficult for patient to open eyes.-   4=Very Severe: eyes are swollen shut.    Erythema:-   0=None.-   1=Mild: slightly dilated blood vessels; color of vessels is    typically pink.-   2=Moderate: more apparent dilation of blood vessels: vessel color is    more intense (redder)-   3=Severe: numerous and obvious dilated blood vessels; the color is    deep red.-   4=Very Severe: large, numerous dilated blood vessels characterized    by severe deep red color.    Itching, as Evaluated by Patient-   0=None.-   1=Mild: an intermittent tickle sensation involving more than just    the corner of the eye.-   2=Moderate: a mild continuous itch (can be localized) without desire    to rub.-   3=Severe: a severe itch with the desire to rub.-   4=Very Severe: an incapacitating itch with an irresistible urge to    rub.

Example 36 Assessment of Allergic Conjunctivitis

According to additional aspects of the present invention, the allergenconcentration established during Visit 1 is used. Evaluation ofconjunctival erythema, edema, and itching is measured before and afterallergen challenge. In order to determine the rapidity of onset,evaluations are done before and after instillation of active eye dropsand vehicle. In one group of 20 subjects, eyes are randomized to receiveeither one drop of active eye drop or one drop of vehicle twice a dayfor two days, and again 10 minutes before allergen challenge. In asecond group of 20 subjects, eyes are randomized to receive either onedrop of active eye drop, or one drop of vehicle, 10 minutes beforeallergen challenge. The allergen determined at Visit 1 or 2 is instilledin both eyes. Erythema, edema, and itching is evaluated at 5 minuteintervals for 20 minutes following instillation of the allergen.

In order to determine the duration of action, evaluations are donebefore and after instillation of active eyedrops and vehicle. In thefirst group of 20 subjects, eyes are randomized to receive either onedrop of active eye drop or one drop of vehicle twice a day for two days,and again 8 hours before allergen challenge. In the second group of 20subjects, eyes are randomized to receive either one drop of active eyedrop, or one drop of vehicle, 8 hours before allergen challenge.Subjects leave the clinic and return 8 hours later. When the subjectreturns, allergen is instilled in both eyes. Erythema, edema, anditching are evaluated at 5 minute intervals for 20 minutes followinginstillation of allergen.

Assessments will be conducted as follows:

Visit 1 (Day 0):

-   Obtain informed consent. Obtain medical, ocular, and allergic    history (to include concomitant medications). If female of    childbearing potential, perform urine pregnancy test. Perform    baseline slit lamp examination. Perform baseline allergen challenge    titrating as needed to obtain positive response (2+ itching and    erythema). Instruct patient regarding post-challenge use of rescue    medications. Schedule patient to return in one week.    Visit 2 (day 7+/−2)-   Update history and query for adverse events. Update medication    history. Perform slit lamp exam. Evaluation of conjunctival    erythema, edema, and itching before and after allergen challenge on    a scale of 0-4. Perform allergen challenge with threshold dose    determined at Visit 1. Randomization: Group I (20 subjects) eyes    will be randomized to receive either one drop of active eye drop or    one drop of vehicle twice a day for two days prior to visit 3.    Instruct Group I subjects regarding use of study eye drops. Instruct    patient regarding post-challenge use of rescue medications. Schedule    patient to return in one week.    Visit 3 (Day 14+/−2)-   Update history and query for adverse events. Update medication    history. Perform slit lamp exam. Evaluation of conjunctival    erythema, edema, and itching before and after instillation of active    eye drops and vehicle on a scale of 0-4. Randomization: Group I: (20    subjects) eyes receive an additional drop of active eye drop or an    additional drop of vehicle 10 minutes before allergen challenge.    Group II: (20 subjects) eyes are randomized to receive either one    drop of active eye drop or one of the vehicle 10 minutes before    challenge. Instill study eyedrops for Groups I and II. Perform    allergen challenge with threshold dose determined at Visits 1 and 2    ten minutes after instillation of study eyedrops. Record findings at    5 minute intervals for 20 minutes. Evaluate which drop was preferred    by the subject. Randomization: Group I (20 subjects) eyes are    randomized to receive either one drop of active eye drop or one drop    of vehicle twice a day for two days prior to visit 4 and again 8    hours prior to allergen challenge. Group II (20 subjects) eyes are    randomized to receive either one drop of active eye drop or one drop    of vehicle 8 hours before allergen challenge. Instruct patient    regarding use of study eye drops. Instruct patient regarding    post-challenge use of rescue medications. Schedule patient to return    in one week.    Visit 4 (Day 21+/−)-   Update history and query for adverse events. Update medication    history. Perform slit lamp exam. Evaluation of conjunctival    erythema, edema, and itching before and after instillation of active    eye drops and vehicle on a scale of 0-4. Perform allergen challenge    with threshold dose determined at Visits 1 and 2 eight hours after    instillation of study eyedrops. Record findings at 5 minute    intervals for 20 minutes. Evaluate which drop was preferred by the    subject. Instruct patient regarding post-challenge use of rescue    medications. Exit patient from study.

The invention claimed is:
 1. A method for treating an irritation,infection or inflammatory eye condition or disease of a subject,comprising administering, by contacting the eye of a subject in needthereof, a therapeutically effective amount of anelectrokinetically-altered ionic aqueous solution of charge-stabilizedoxygen-containing nanobubbles having an average diameter of less than100 nanometers and stably configured in the ionic aqueous solution in anamount sufficient for treating an irritation, infection or inflammatoryeye condition or disease, or at least one symptom thereof.
 2. The methodof claim 1, wherein the charge-stabilized oxygen-containing nanobubblesare the major charge-stabilized gas-containing nanobubble species in thesolution.
 3. The method of claim 1, wherein the percentage of dissolvedoxygen molecules present in the solution as the charge-stabilizedoxygen-containing nanobubbles is greater than 0.01%.
 4. The method ofclaim 1, wherein the total dissolved oxygen is substantially present inthe charge-stabilized oxygen-containing nanobubble.
 5. The method ofclaim 1, wherein the charge-stabilized oxygen-containing nanobubbleshave an average diameter of less than 90 nm.
 6. The method of claim 1,wherein the ionic aqueous solution comprises a saline solution.
 7. Themethod of claim 1, wherein the solution is superoxygenated.
 8. Themethod of claim 1, wherein the solution comprises a form of solvatedelectrons.
 9. The method of claim 1, wherein alteration of theelectrokinetically altered aqueous solution comprises exposure of thefluid solution to hydrodynamically-induced, localized electrokineticeffects.
 10. The method of claim 9, wherein, exposure to the localizedelectrokinetic effects comprises exposure to at least one of voltagepulses and current pulses.
 11. The method of claim 9, wherein theexposure of the solution to hydrodynamically-induced, localizedelectrokinetic effects, comprises exposure of the solution toelectrokinetic effect-inducing structural features of a device used togenerate the fluid.
 12. The method of claim 1, wherein the irritation,infection or inflammatory eye condition or disease comprises at leastone selected from the group consisting of dry eye, allergy irritation,corneal irritation, bacterial infection, and cellular damage.
 13. Themethod of claim 12, wherein the irritation, infection or inflammatoryeye condition or disease eye condition or disease comprises at least oneof dry eye, allergy irritation, and corneal irritation.
 14. The methodof claim 13, wherein the irritation, infection or inflammatory eyecondition or disease eye condition or disease comprises dry eye orallergy irritation.
 15. The method of claim 1, wherein the at least onesymptom of the irritation, infection or inflammatory eye condition ordisease is related to at least one condition selected from the groupconsisting of: dry eye-related irritation, dry eye-related cellulardamage, and allergic inflammation.
 16. The method of claim 1, whereinthe electrokinetically altered aqueous solution modulates localized orcellular levels of nitric oxide.
 17. The method of claim 1 wherein theelectrokinetically altered aqueous solution promotes a localizeddecrease at the site of administration of at least one cytokine selectedfrom the group consisting of: IL-1-beta, IL-8, TNF-alpha, and TNF-beta.18. The method of claim 1, further comprising a synergistic ornon-synergistic inhibition or reduction in inflammation bysimultaneously or adjunctively treating the subject with anotheranti-inflammatory or eye treatment agent.
 19. The method of claim 18,wherein said other anti-inflammatory agent comprises a steroid orglucocorticoid steroid.
 20. The method of claim 19, wherein theglucocorticoid steroid comprises Budesonide or an active derivativethereof.
 21. The method of claim 1, comprising combination therapy,wherein at least one additional therapeutic agent is administered to thepatient.
 22. The method of claim 21, wherein, the at least oneadditional therapeutic agent is selected from the group consisting of:anti-microbial agent, anti-inflammatory agent, pain reliever,anesthetic, vitamin, cytokine, adjuvant, preservative, salt, MMPinhibitors, short-acting β₂-agonists, long-acting β₂-agonists,anticholinergics, corticosteroids, systemic corticosteroids, mast cellstabilizers, leukotriene modifiers, methylxanthines, β₂-agonists,albuterol, levalbuterol, pirbuterol, artformoterol, formoterol,salmeterol, anticholinergics including ipratropium and tiotropium;corticosteroids including beclomethasone, budesonide, flunisolide,fluticasone, mometasone, triamcinolone, methyprednisolone, prednisolone,prednisone; leukotriene modifiers including montelukast, zafirlukast,and zileuton; mast cell stablizers including cromolyn and nedocromil;methylxanthines including theophylline; combination drugs includingipratropium and albuterol, fluticasone and salmeterol, budesonide andformoterol; antihistamines including hydroxyzine, diphenhydramine,loratadine, cetirizine, and hydrocortisone; immune system modulatingdrugs including tacrolimus and pimecrolimus; cyclosporine; azathioprine;mycophenolatemofetil; and combinations thereof.
 23. The method of claim21, wherein the at least one additional therapeutic agent is a TSLPand/or TSLPR antagonist.
 24. The method of claim 23, wherein the TSLPand/or TSLPR antagonist is selected from the group consisting ofneutralizing antibodies specific for TSLP and the TSLP receptor, solubleTSLP receptor molecules, and TSLP receptor fusion proteins, includingTSLPR-immunoglobulin Fc molecules or polypeptides that encode componentsof more than one receptor chain.
 25. The method of claim 1, wherein thecharge-stabilized oxygen-containing nanobubbles are stably configured inthe ionic aqueous solution in an amount sufficient to provide, uponcontact of a living cell by the fluid, modulation of at least one ofcellular membrane potential and cellular membrane conductivity.
 26. Themethod of claim 25, wherein the membrane associated protein comprises atleast one selected from the group consisting of receptors, transmembranereceptors, ion channel proteins, intracellular attachment proteins,cellular adhesion proteins, integrins, etc.
 27. The method of claim 26,wherein the transmembrane receptor comprises a G-Protein CoupledReceptor (GPCR).
 28. The method of claim 27, wherein the G-ProteinCoupled Receptor (GPCR) interacts with a G protein α subunit.
 29. Themethod of claim 28, wherein the G protein α subunit comprises at leastone selected from the group consisting of Gα_(s), Gα_(i), Gα_(q), andGα₁₂.
 30. The method of claim 29, wherein the at least one G protein αsubunit is Gα_(q).
 31. The method of claim 25, wherein modulating atleast one of cellular membrane potential and cellular membraneconductivity, comprises modulating whole-cell conductance.
 32. Themethod of claim 31, wherein modulating whole-cell conductance, comprisesmodulating at least one linear or non-linear voltage-dependentcontribution of the whole-cell conductance.
 33. The method of claim 25,wherein modulation of at least one of cellular membrane potential andcellular membrane conductivity comprises modulation of acalcium-dependent cellular messaging pathway or system.
 34. The methodof claim 25, wherein modulation of at least one of cellular membranepotential and cellular membrane conductivity comprises modulation ofphospholipase C activity.
 35. The method of claim 25, wherein modulationof at least one of cellular membrane potential and cellular membraneconductivity comprises modulation of adenylate cyclase (AC) activity.36. The method of claim 25, wherein modulation of at least one ofcellular membrane potential and cellular membrane conductivity comprisesmodulation of intracellular signal transduction associated with at leastone condition or symptom selected from the group consisting of: dry eye,allergy irritation, corneal irritation, bacterial infection, andcellular damage.
 37. The method of claim 1, comprising administration ofthe electrokinetically-altered aqueous solution to a cell network orlayer, and further comprising modulation of an intercellular junctiontherein.
 38. The method of claim 37, wherein the intracellular junctioncomprises at least one selected from the group consisting of tightjunctions, gap junctions, zona adherins and desmasomes.
 39. The methodof claim 37, wherein the cell network or layers comprises at least oneselected from the group consisting of corneal epithelial layer andcorneal endothelium.
 40. The method of claim 1, wherein theelectrokinetically-altered aqueous solution is oxygenated, and whereinthe oxygen in the fluid is present in an amount of at least 8 ppm oxygenat atmospheric pressure.
 41. The method of claim 1, wherein theelectrokinetically altered aqueous solution comprises at least one of aform of solvated electrons, and electrokinetically modified or chargedoxygen species.
 42. The method of claim 41, wherein the form of solvatedelectrons or electrokinetically modified or charged oxygen species arepresent in an amount of at least 0.01 ppm.
 43. The method of claim 42,wherein the electrokinetically-altered aqueous solution comprises a formof solvated electrons stabilized, at least in part, by molecular oxygen.44. The method of claim 25, wherein the ability to alter at least one ofcellular membrane potential and cellular membrane conductivity persistsfor at least two months in a closed gas-tight container.
 45. The methodaccording to claim 1, wherein the electrokinetically-altered aqueoussolution comprises a contact lens solution or multi-use contact lenssolution.
 46. The method according to claim 45, wherein the contact lenssolution or multi-use contact lens solution comprises a storagesolution, a wetting solution, or a cleaning solution.
 47. The methodaccording to claim 12, wherein the electrokinetically-altered aqueoussolution inhibits the growth of Pseudomonas bacteria.
 48. The methodaccording to claim 1, wherein the irritation, infection or inflammatoryeye condition is the result of a wound.
 49. The method according toclaim 48, wherein the wound is selected from the group consisting of:lacerations, abrasions, rupture, puncture wounds, chemical, thermal, orradiation-induced burns, cuts, scrapes, incisions, blisters, ulcers, andsurgical wounds.
 50. The method according to claim 49, wherein thesurgical wound is a result of at least one surgery selected from thegroup consisting of: laser keratotomy, cataract removal, lensimplantation or removal, corneal alterations, laser-assisted in situkeratomileusis (LASIK), intraLASIK, extracapsular surgery,phacoemulsification, vitreoretinal surgery, glaucoma procedures,neuro-ophthalmic surgery, and strabismus surgery.
 51. The method ofclaim 12, wherein the irritation, infection or inflammatory eyecondition or disease comprises dry eye, and wherein at least one sign orsymptom selected from the group consisting of: decrease in aqueous tearproduction, increase in tear evaporation, and increase in cellularinflammation is ameliorated.
 52. The method according to claim 51,wherein said ameliorating is assessed by at least one measurementselected from the group consisting of corneal staining, conjunctivalstaining, tear volume, bulbar conjunctival erythema, tear film height,ocular surface healing, level of infection, level of inflammation, levelof ocular surface damage, and level of inferior tear meniscus.
 53. Themethod according to claim 1, wherein said contacting comprises topicallyapplying said fluid to said eye.
 54. The method according to claim 1,wherein said treatment comprises an adjunct therapy and said subjectfurther undergoes a primary treatment for an eye condition.
 55. Themethod according to claim 54, wherein said primary treatment comprisesadministering to said subject at least one medication selected from thegroup consisting of artificial tears, anti-inflammatory medications,steroids, antihistamines, antibiotics, and vasoconstrictor medications.56. The method according to claim 55, wherein said administeringcomprises topically or orally administering said medication to thesubject.
 57. The method according to claim 1, wherein said contactingcomprises topically or systemically contacting said eye of the subject.58. The method according to claim 52, wherein said treatment results ina reduction in corneal or conjuctival staining.
 59. The method accordingto claim 52, wherein said treatment results in an increase in tearvolume or tear film height.
 60. The method of claim 25, whereinmodulation of at least one of cellular membrane potential and cellularmembrane conductivity comprises modulating at least one of cellularmembrane structure or function comprising altering of at least one of aconformation, ligand binding activity, or a catalytic activity of amembrane associated protein.
 61. The method of claim 1, wherein theamount of oxygen present in charge-stabilized oxygen-containingnanobubbles of the electrokinetically-altered solution is at least 8 ppmoxygen at atmospheric pressure.
 62. The method of claim 1, wherein amajority of the charge-stabilized oxygen-containing nanobubbles have adiameter of less than 100 nanometers.
 63. The method of claim 22,wherein the MMP inhibitor comprises an inhibitor of MMP-9 and/or andinhibitor of MMP-2.